Kit and detection method for detecting epidemic strain of internally-running sheep virus

Abstract

The invention discloses a kit and a detection method for detecting an internally-running sheep virus epidemic strain, the kit comprises a crRNA sequence and an RT-RPA primer, the detection method comprises an RT-RPA reaction system and a CRISPR / cas12a reaction system, RT-RPA nucleic acid amplification and CRISPR / cas12a nucleic acid detection are combined, a two-step detection method is adopted, firstly, RT-RPA nucleic acid amplification is carried out, secondly, RT-RPA nucleic acid amplification is carried out, thirdly, RT-RPA nucleic acid amplification is carried out, secondly, CRISPR / cas12a nucleic acid detection is carried out, and thirdly, RT-RPA nucleic acid detection is carried out. Then, the RT-RPA nucleic acid product is added into a CRISPR / cas12a nucleic acid detection system, and the Chinese epidemic strain of the internally-running sheep virus can be detected. The detection method is simple, efficient, low in dependence on instruments and equipment, good in sensitivity, specificity and stability and good in social application prospect.

Description

Technical Field

[0001] The invention relates to the field of kit preparation, and in particular to a kit and a method for detecting Nairobi sheep virus epidemic strains. Background Art

[0002] Nairobi sheep disease (NSD) is a zoonotic tick-borne infectious disease caused by the Nairobi sheep virus (NSDV epidemic strain). The NSDV epidemic strain can infect sheep and goats, causing acute hemorrhagic gastroenteritis in sheep and goats, with a mortality rate of up to 90%; it can also infect humans, causing mild flu-like symptoms such as fever, nausea, and vomiting. In 2015 and 2019, it was detected in tick samples in Northeast my country and Hubei Province, respectively. In 2022, the NSDV epidemic strain was detected in sheep flocks in Henan for the first time in my country. The NSDV epidemic strain has posed a serious threat to my country's sheep and goat farming industry.

[0003] There is currently no effective treatment for NSD, so early detection and early diagnosis are of great significance for the prevention and control of NSD. The existing detection methods for NSDV epidemic strains are divided into two categories: molecular detection and serological detection. The existing molecular detection method for NSDV epidemic strains is mainly based on RT-PCR technology. This method has the advantages of high sensitivity and high specificity. It is also the standard method for detecting NSDV epidemic strains recommended by OIE, but it relies on instruments and professional technicians; while the existing serological detection methods such as ELISA are simple to operate, but there are generally serious cross-reactions, which can easily lead to false positive results and poor specificity. In summary, the existing detection methods cannot take into account the advantages of high sensitivity, high specificity, rapidity, and portability, and are difficult to promote for on-site detection.

[0004] The detection technology based on the CRISPR-Cas system is a new detection technology developed in recent years and has been widely used in the nucleic acid detection of pathogens. The CRISPR-Cas system consists of crRNA and Cas protein. After crRNA specifically recognizes and binds to the target, the conformation of the Cas protein changes and its cleavage activity is activated. It can not only cleave the target, but also indiscriminately cleave the fluorescent reporter molecule. After the fluorescent reporter molecule is cleaved, it releases fluorescence and undergoes at least 10 4 The signal is amplified by 100 times, thus visualizing the test results. Compared with the traditional PCR-based detection technology, the nucleic acid detection technology based on the CRISPR-Cas system is convenient, fast, low-cost, and does not require expensive equipment and professionals. It has the potential to be developed into an on-site detection tool. Summary of the invention

[0005] Based on this, in order to overcome the defects and shortcomings of the existing early detection and prevention and control technology of Nairobi sheep virus (NSDV epidemic strain) infection, the purpose of the present invention is to provide a kit and a detection method for detecting the Nairobi sheep virus epidemic strain, which is a rapid on-site NSDV epidemic strain detection method with high sensitivity, strong specificity, low instrument dependence, and quick and simple operation, in order to address the shortcomings of the existing NSDV epidemic strain detection technology, such as poor specificity, long time consumption, high dependence on instruments, and the need for professional personnel to operate and cannot meet the requirements of rapid on-site detection.

[0006] The present invention provides a kit and a method for detecting the epidemic strain of Nairobi sheep virus. The specific technical scheme is as follows:

[0007] A kit for detecting the epidemic strain of Nairobi sheep virus, the kit comprising a crRNA sequence and an RT-RPA primer;

[0008] Wherein, the crRNA sequence is: SEQ ID NO.1-8; or a nucleotide sequence with two bases therefrom.

[0009] Further, the RT-RPA primers are: F3 (SEQ ID NO.15) / R3 (SEQ ID NO.16), F3 (SEQ ID NO.15) / R4 (SEQ ID NO.18), F4 (SEQ ID NO.17) / R3 (SEQ ID NO.16), F4 (SEQ ID NO.17) / R4 (SEQ ID NO.18), F5 (SEQ ID NO.19) / R5 (SEQ ID NO.20), F5 (SEQ ID NO.19) / R6 (SEQ ID NO.22), F6 (SEQ ID NO.21) / R5 (SEQ ID NO.20), F6 (SEQ ID NO.21) / R6 (SEQ ID NO.22); or a nucleotide sequence pair that differs from it by no more than 2 bases.

[0010] Furthermore, the kit also includes Cas12a protein and single-stranded DNA marked with a fluorescent signal.

[0011] Furthermore, the single-stranded DNA labeled with a fluorescent signal is 5′-FAM-TTATT-BHQ-3′.

[0012] Furthermore, the kit also includes C buffer, L buffer, B buffer, and PR-core.

[0013] Furthermore, the crRNA sequence is designed and prepared within a target fragment by analyzing and selecting a suitable target, and the target fragment is obtained by performing specificity and conservation analysis on the sequence of the NSDV epidemic strain using BLAST and MEGA software.

[0014] A kit is used for a detection method for non-disease diagnosis purposes of Nairobi sheep virus, the detection method comprising RT-RPA reaction and CRISPR / Cas12a reaction.

[0015] Furthermore, the temperature of the RT-RPA reaction is 42°C, and the temperature of the CRISPR / Cas12a reaction is 37°C.

[0016] Compared with the prior art, the beneficial effects include:

[0017] 1. The present invention provides a crRNA and RT-RPA primer pair for detecting NSDV epidemic strains, wherein the crRNA has good specificity, and the RT-RPA primer pair has high amplification efficiency, both of which have high sensitivity and accuracy for detection. The present invention also performs a joint screening of crRNA and RT-RPA primers, further improving the sensitivity and accuracy of the method of the present invention, and a minimum of 50 copies of target RNA can be detected.

[0018] 2. The detection method of the present invention is low-cost, does not require a professional and standardized laboratory, and does not require the use of large-scale instruments and equipment (fluorescence quantitative PCR instrument, etc.). It only requires incubation in a water bath or a constant temperature incubator, or even incubation at body temperature, and a fluorescence detector. This not only reduces the detection cost, but is also more suitable for first-line detection.

[0019] 3. The detection time of the present invention is short. The current RT-PCR reaction time is about 1.5 hours, while the method established by the present invention first requires 30 minutes for constant temperature amplification, and then only 15 minutes for fluorescence detection and observation, a total of about 45 minutes. Therefore, the detection method established by the present invention is more conducive to the early rapid detection of NSDV epidemic strains, which is very important for the early diagnosis and prevention and control of NSDV epidemic strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention can be further understood from the following description in conjunction with the accompanying drawings. The components in the figures are not necessarily drawn to scale, but the emphasis is placed on illustrating the principles of the embodiments. In different views, the same reference numerals designate corresponding parts.

[0021] Figure 1 The double-stranded DNA reverse transcribed from the NSDV epidemic strain was used as the target, and the fluorescence value was measured by CRISPR / Cas12a for 30 minutes when 8 crRNAs were screened.

[0022] Figure 2 The agarose gel electrophoresis results of RT-RPA amplification for 30 min when 12 RT-RPA primer pairs were screened.

[0023] Figure 3 The NSDV epidemic strain RNA standard was used as a target after multiple dilution. After RT-RPA amplification for 30 minutes using an efficient RT-RPA primer pair (F5 / R6), CRISPR / Cas12a detection was performed for 30 minutes using efficient crRNA and the amplified product. The sensitivity fluorescence curve analysis results and the blue light irradiation results of the reaction tube were shown.

[0024] Figure 4 AB is the sensitivity fluorescence curve analysis results of using the NSDV epidemic strain RNA standard as the target after multiple dilution, using the high-efficiency RT-RPA primer pair (F4 / R4) for RT-RPA amplification for 30 minutes, and using the high-efficiency crRNA and the amplified product for CRISPR / Cas12a detection for 30 minutes, as well as the blue light irradiation results of the reaction tube.

[0025] Figure 5 AD is the fluorescence curve results of the analysis of the stability of the NSDV epidemic strain RNA standard using the RT-RPA-CRISPR / Cas12a method and the blue light irradiation results of the reaction tube.

[0026] Figure 6 This is the agarose gel electrophoresis result of clinical sample testing using the RT-RPA method.

[0027] Figure 7 AC is the fluorescence curve result of clinical samples detected at 30 minutes using the constructed RT-RPA-CRISPR / Cas12a method, the terminal fluorescence bar graph analysis result, and the blue light irradiation result of the reaction tube. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0030] A kit for detecting the epidemic strain of Nairobi sheep virus in one embodiment of the present invention, the kit comprising a crRNA sequence and an RT-RPA primer;

[0031] Wherein, the crRNA sequence is: SEQ ID NO.1-8; or a nucleotide sequence with two bases therefrom.

[0032] In one embodiment, the RT-RPA primers are: F3 (SEQ ID NO.15) / R3 (SEQ ID NO.16), F3 (SEQ ID NO.15) / R4 (SEQ ID NO.18), F4 (SEQ ID NO.17) / R3 (SEQ ID NO.16), F4 (SEQ ID NO.17) / R4 (SEQ ID NO.18), F5 (SEQ ID NO.19) / R5 (SEQ ID NO.20), F5 (SEQ ID NO.19) / R6 (SEQ ID NO.22), F6 (SEQ ID NO.21) / R5 (SEQ ID NO.20), F6 (SEQ ID NO.21) / R6 (SEQ ID NO.22); or a nucleotide sequence pair that differs from it by no more than 2 bases.

[0033] In one embodiment, the kit also includes Cas12a protein and single-stranded DNA labeled with a fluorescent signal.

[0034] In one embodiment, the single-stranded DNA labeled with a fluorescent signal is 5′-FAM-TTATT-BHQ-3′.

[0035] In one embodiment, the kit further includes C buffer, L buffer, B buffer, and PR-core.

[0036] In one of the embodiments, the crRNA sequence is designed and prepared within a target fragment by analyzing and selecting a suitable target, and the target fragment is obtained by performing specificity and conservation analysis on the sequence of the NSDV epidemic strain using BLAST and MEGA software.

[0037] A kit is used for a detection method for non-disease diagnosis purposes of Nairobi sheep virus, the detection method comprising RT-RPA reaction and CRISPR / Cas12a reaction.

[0038] In one embodiment, the temperature of the RT-RPA reaction is 42°C, and the temperature of the CRISPR / Cas12a reaction is 37°C.

[0039] The test kit in the above scheme has high sensitivity for detecting NSDV epidemic strains, and has better detection efficiency and detection effect, and plays a positive response role in the early diagnosis and prevention and control of NSDV epidemic strains.

[0040] The embodiments of the present invention will be described in detail below with reference to specific examples.

[0041] Embodiment 1:

[0042] Synthesis of double-stranded DNA templates targeting NSDV epidemic strains.

[0043] In order to establish an RT-RPA-CRISPR / Cas12a detection method for NSDV epidemic strains, the present invention selects the target region for detection through specificity and conservation analysis, synthesizes a target double-stranded DNA template for NSDV epidemic strain detection containing a T7 promoter, which is used for the screening of efficient crRNA and the transcription of target RNA. The specific sequence is shown in SEQ ID NO.1.

[0044] Embodiment 2:

[0045] Synthesis of target RNA standards of NSDV epidemic strains.

[0046] The NSDV epidemic strain target RNA standard was transcribed using the double-stranded DNA template of the NSDV epidemic strain target, and the specific sequence is shown in SEQ ID NO.2.

[0047] Embodiment 3:

[0048] Design and synthesis of crRNA.

[0049] The quality of crRNA is directly related to the sensitivity and accuracy of the detection method. In order to screen crRNA with higher efficiency, 8 crRNAs were designed for the target domain of NSDV epidemic strains, and their sequences are shown in Table 1.

[0050] Table 1: crRNA

[0051]

[0052] The synthesis method of 8 crRNAs is as follows:

[0053] Two complementary primers were used to synthesize the DNA template of the crRNA sequence, and the PCR reaction system was as shown in Table 2.

[0054] Table 2: PCR reaction system

[0055]

[0056] Add the components in Table 2 above to a 200 μL centrifuge tube, mix well and centrifuge. Place in a PCR instrument for reaction:

[0057] Pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s, annealing at 55°C for 20 s, extension at 72°C for 4 s, repeated 33 cycles; and finally extension at 72°C for 3 min to obtain a double-stranded DNA template containing a T7 promoter for crRNA. PCR primers were synthesized by Qingke Biotechnology, and DNA polymerase was purchased from Novazonics.

[0058] The DNA template was purified by nucleic acid co-precipitation and its concentration was measured on Qubit 3.0. The purified DNA template was stored in a -20°C refrigerator.

[0059] The crRNA was transcribed in vitro using the full-form gold T7 transcription kit, and the transcription system is shown in Table 3.

[0060] Table 3: Transcription system

[0061]

[0062] Add the components in Table 3 to a 1.5 mL centrifuge tube, flick to mix, and centrifuge. Incubate overnight in a 37°C incubator; then add 45 μL of DEPC water and 2 μL of DNase I, and incubate at 37°C for 30 min to obtain crRNA.

[0063] RNA was purified using the Trizol method, and its concentration was measured on Qubit 3.0. The purified crRNA was stored in a -80°C refrigerator.

[0064] Embodiment 4:

[0065] Screening of efficient RT-RPA primers.

[0066] The primers designed for RT-RPA amplification, F (forward) / R (reverse), have sequences shown in Table 4.

[0067] Table 4: Primers for RT-RPA amplification

[0068]

[0069] RT-RPA amplification was performed using the RT-RPA primers in Table 4 above. The RT-RPA system is shown in Table 5.

[0070] Table 5: RT-RPA system

[0071]

[0072]

[0073] Use RNA Constant Temperature Rapid Amplification Kit (Liquid-based) to prepare the premix system, mix well and dispense into 200μL PCR tubes, add the corresponding primer pair combinations, and amplify at 42℃ for 30min. Then load 2% agarose gel, observe the position and brightness of the bands, and select 2 pairs of efficient RPA primers. The results of agarose gel nucleic acid electrophoresis at 30min amplification are as follows: Figure 2 As shown in the figure, NC represents negative control, PC represents positive control, M represents Marker, and the circled part represents the RT-RPA primer pair with correct band size and high amplification efficiency, which are: F4 (SEQ ID NO.17) / R4 (SEQ ID NO.18); F5 (SEQ ID NO.19) / R6 (SEQ ID NO.22)

[0074] Embodiment 5:

[0075] Screening of efficient crRNA.

[0076] Using the double-stranded DNA template of the NSDV epidemic strain target as the target, the crRNA with the highest efficiency located in the amplified fragment of the two pairs of efficient RT-RPA primers was screened from the 8 crRNAs prepared above, the reaction time was 30 minutes, and the real-time fluorescence value was read using the Suzhou Crui constant temperature amplification instrument. The reaction system is shown in Table 6.

[0077] Table 6: Reaction system

[0078]

[0079]

[0080] The results are as follows Figure 1 As shown: at a reaction time of 30 minutes, the fluorescence curve of crRNA2 located in the F4 / R4 amplification region is the highest; the fluorescence curve of crRNA7 located in the F5 / R6 amplification region is the highest, indicating that crRNA2 and crRNA7 are the most efficient crRNAs in the two amplification regions, respectively.

[0081] Embodiment 6:

[0082] Establishment of the RT-RPA-CRISPR / Cas12a method for detecting NSDV epidemic strains.

[0083] The F4 / R4 primer pair and the F5 / R6 primer pair were used for RT-RPA reaction, and the system is shown in Table 7.

[0084] Table 7: RT-RPA reaction system

[0085]

[0086] Take the above RT-RPA product and add it to the Cas12a reaction system. The reaction system is as shown in Table 8.

[0087] Table 8: Cas12a reaction system

[0088]

[0089]

[0090] Embodiment 7:

[0091] Sensitivity analysis of RT-RPA-CRISPR / Cas12a for detecting NSDV.

[0092] The NSDV target RNA standard with a known copy number was diluted to 0 / 10 / 100 / 500 / 1000 copies, and the reaction was carried out according to the method and conditions in Example 6. The RT-RPA primers in the RT-RPA reaction system were respectively the high-efficiency primer pairs (F4 / R4) and (F5 / R6), and the templates were respectively the above-mentioned NSDV RNA standards with different copies; the Cas12a reaction system used high-efficiency primer pairs to amplify the corresponding high-efficiency crRNA (F4 / R4-crRNA2) and (F5 / R6-crRNA7) in the region. The fluorescence value was read using Suzhou Krui constant temperature amplification instrument.

[0093] The results are as follows Figure 3 and Figure 4 As shown, it can be seen that the detection performance of (F4 / R4-crRNA2) is much stronger than that of (F5 / R6-crRNA7), and the system of the present invention can detect a minimum of 10 copies of the sample, indicating that it has excellent detection sensitivity and has good application prospects in early detection of infection.

[0094] Embodiment 8:

[0095] Stability analysis of RT-RPA-CRISPR / Cas12a detection of NSDV epidemic strains.

[0096] Three independent RT-RPA-CRISPR / Cas12a detection experiments were performed using 50 copies of the NSDV epidemic strain target RNA standard. Stability analysis was performed according to the RT-RPA-CRISPR / Cas12a detection method and conditions in Example 6, and the fluorescence value was read using Suzhou Crui Constant Temperature Amplifier.

[0097] The results are as follows Figure 5As shown: the fluorescence of the control group of the three experiments was maintained at an extremely low level within 30 minutes of the reaction, and there was no obvious upward trend, while the fluorescence of the experimental group had an obvious upward trend within 30 minutes, and the fluorescence values ​​of the three experimental groups were relatively close at 30 minutes. The above results show that the method for detecting Toxoplasma gondii nucleic acid constructed by the present invention has strong stability.

[0098] Embodiment 9:

[0099] Analysis of the accuracy of RT-RPA-CRISPR / Cas12a in detecting NSDV epidemic strains.

[0100] The RT-RPA-CRISPR / Cas12a detection method in Example 6 was used to detect 13 clinical samples to be tested, and compared with the RT-PCR detection method of NSDV currently recommended by OIE to verify the accuracy of the detection method of the present invention.

[0101] The results are as follows Figure 6 , Figure 7 shown. Figure 6 This is the agarose gel electrophoresis diagram of clinical samples detected by RT-PCR. Figure 7 AC are the fluorescence curve results, terminal fluorescence bar graph analysis results and reaction tube blue light irradiation results of the clinical samples detected by the constructed RT-RPA-CRISPR / Cas12a method for 30 minutes, respectively. Samples 1 and 6 were negative in the two detection methods, and the remaining samples were positive. The RT-RPA-CRISPR / Cas12a detection method of the present invention is consistent with the RT-PCR results, that is, the method of the present invention is accurate and effective.

[0102] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0103] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A kit for detecting the epidemic strain of Nairobi sheep virus, characterized in that: The kit includes a crRNA sequence and a RT-RPA primer; Wherein, the crRNA sequence is: SEQ ID NO.1-8; or a nucleotide sequence with two bases therefrom.

2. The kit according to claim 1, characterized in that The RT-RPA primers are: F3 (SEQ ID NO.15) / R3 (SEQ ID NO.16), F3 (SEQ ID NO.15) / R4 (SEQ ID NO.18), F4 (SEQ ID NO.17) / R3 (SEQ ID NO.16), F4 (SEQ ID NO.17) / R4 (SEQ ID NO.18), F5 (SEQ ID NO.19) / R5 (SEQ ID NO.20), F5 (SEQ ID NO.19) / R6 (SEQ ID NO.22), F6 (SEQ ID NO.21) / R5 (SEQ ID NO.20), F6 (SEQ ID NO.21) / R6 (SEQ ID NO.22); or a nucleotide sequence pair that differs from it by no more than 2 bases.

3. The kit according to claim 2, characterized in that The kit also includes Cas12a protein and single-stranded DNA marked with a fluorescent signal.

4. The kit according to claim 3, characterized in that The single-stranded DNA with a fluorescent signal marker is 5′-FAM-TTATT-BHQ-3′.

5. The kit according to claim 4, characterized in that The kit also includes Cbuffer, L buffer, Bbuffer, and PR-core.

6. The kit according to claim 1, characterized in that The crRNA sequence is designed and prepared within a target fragment by analyzing and selecting a suitable target, and the target fragment is obtained by performing specificity and conservation analysis on the sequence of the NSDV epidemic strain using BLAST and MEGA software.

7. The kit according to claim 1 is used for a detection method for non-disease diagnosis purposes of Nairobi sheep virus, wherein the detection method comprises an RT-RPA reaction and a CRISPR / Cas12a reaction.

8. The kit according to claim 7, characterized in that The temperature of the RT-RPA reaction was 42°C, and the temperature of the CRISPR / Cas12a reaction was 37°C.

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