Rapid detection method for orf virus based on MIRA technology

Abstract

The invention relates to the technical field of orf virus detection, and discloses a rapid orf virus detection method based on an MIRA technology, and the method comprises the following steps: step 1, extracting nucleic acid from a sample to be detected; 2, carrying out isothermal amplification by taking the extracted nucleic acid as a template and adopting an MIRA reaction system; 3, the MIRA reaction system comprises a specific primer pair aiming at the ORFVB2L gene, and the sequence of the specific primer pair is shown as SEQ ID NO: 1 (F2) and SEQ ID NO: 2 (R3). The sequence of the fluorescent probe is as shown in SEQ ID NO: 3 (P1). According to the invention, high-sensitivity, high-specificity, rapid, low-cost and on-site detection of the orf virus is realized through the MIRA technology, the method is obviously superior to a traditional method, and the method is suitable for basic livestock breeding, epidemic disease monitoring and emergency diagnosis of sudden epidemic situations.

Description

Technical Field

[0001] This invention relates to the field of sheep poxvirus detection technology, and in particular to a rapid detection method for sheep poxvirus based on MIRA technology. Background Technology

[0002] Oral pustulosis, also known as contagious pustular eczema in sheep, is an acute, contagious zoonotic disease caused by the orfvirus (ORFV). It can infect sheep and goats and seriously endangers the development of sheep farming.

[0003] Yunnan Province, located in southwestern China, possesses a unique geographical location and significant strategic importance. ORFV (oral thrush) has long been a problem in sheep flocks, but there are currently no reports of the disease in Yunnan Province. Furthermore, existing detection methods are mostly laboratory-based, complex, time-consuming, and expensive, failing to provide rapid detection. Therefore, there is an urgent need to rapidly improve diagnostic and detection methods for ORFV.

[0004] This study aims to investigate the ORFV infection status in some areas of Yunnan Province and develop a rapid detection method for ORFV using the MIRA assay to meet the needs of first-line clinical rapid initial screening for ORFV. The investigation of ORFV infection status in Yunnan Province will provide new insights into the transmission and control of ORFV. Summary of the Invention

[0005] To address the technical problems mentioned in the background section, this invention provides a rapid detection method for sheep poxvirus based on MIRA technology.

[0006] This invention is achieved using the following technical solution: a rapid detection method for sheep poxvirus based on MIRA technology, the method comprising the following steps:

[0007] Step 1: Extract nucleic acid from the sample to be tested;

[0008] Step 2: Using the extracted nucleic acid as a template, perform isothermal amplification using the MIRA reaction system;

[0009] Step 3, wherein the MIRA reaction system comprises:

[0010] The specific primer pairs targeting the ORFVB2L gene are shown in SEQ ID NO:1(F2) and SEQ ID NO:2(R3).

[0011] The fluorescent probe has the sequence shown in SEQ ID NO:3(P1);

[0012] And MIRA basic reaction reagents;

[0013] Step 4: React at a constant temperature of 42℃ for 5-20 minutes, and judge the results by real-time fluorescence detection.

[0014] Furthermore, the MIRA reaction system is preheated for 5 minutes before the reaction.

[0015] Furthermore, the probe is a FAM / BHQ1 dual-labeled probe, with a tetrahydrofuran (THF) cleavage site at the 35th nucleotide position at its 5' end.

[0016] Furthermore, the lowest detection limit of the method is 7.63 × 10⁻⁶. 9 copies / μL.

[0017] Furthermore, the sample to be tested is a sheep nasal swab, serum, or lesion tissue.

[0018] The second objective of this invention is to provide a MIRA kit for detecting sheep poxvirus, the kit comprising:

[0019] The specific primer pairs described are SEQ ID NO:1 and SEQ ID NO:2;

[0020] The fluorescent probe described is SEQ ID NO:3;

[0021] The basic reaction reagents for MIRA include recombinase, DNA polymerase, single-stranded binding protein, dNTPs, buffer, and magnesium ions.

[0022] Furthermore, the kit is suitable for isothermal reactions at 42°C and does not require a thermal cycler.

[0023] The third objective of this invention is to propose the application of the above-mentioned method or kit in the rapid on-site diagnosis of poxvirus.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This invention discloses a rapid detection method and kit for sheep pox virus based on MIRA technology. Its beneficial technical effects are mainly reflected in four aspects: "faster, more accurate, simpler, and wider application".

[0026] First, the reaction system can complete amplification in just 5–20 minutes under constant temperature of 42℃. With a 5-minute preheating step, the entire process takes less than half an hour, which is significantly faster than the 2–3 hour detection cycle of traditional PCR. At the same time, the specific primer pair F2 / R3 and the FAM / BHQ1 double-labeled probe P1 designed for the ORFVB2L gene showed no amplification signal against seven common pathogens, including BTV, PPRV, PRV, FPV, MG, and MS, in a large number of cross-tests, demonstrating excellent specificity and ensuring reliable results.

[0027] Secondly, the method has extremely high sensitivity, enabling accurate screening of early, low-load samples. In 62 clinical swab samples randomly collected in Qujing, Lijiang, Baoshan and other places in Yunnan, the positive detection rate of this method reached 67.7%, while that of PCR was only 18.5% during the same period, demonstrating its outstanding advantages in field application.

[0028] Secondly, the entire detection process does not require a thermal cycler; a constant-temperature metal bath or even a water bath is sufficient. The accompanying kit pre-packages recombinant enzymes, polymerases, primers, probes, and buffer systems. Users only need to add the template to start the test, significantly reducing dependence on experimental sites and equipment. This truly meets the rapid on-site diagnostic needs of farms, border ports, and grassroots veterinary stations. Furthermore, it offers strong sample type compatibility; sheep nasal swabs, serum, and lesion tissues can all be directly used for nucleic acid extraction and detection, eliminating cumbersome pretreatment. In terms of economic cost, the reaction cost per kit is more than 40% lower than conventional PCR, and it can be transported and stored at room temperature for short periods, significantly reducing cold chain logistics expenses.

[0029] Finally, this invention fills the technological gap in the rapid detection of orofaculum virus in sheep in Yunnan Province, and provides a powerful tool for active monitoring, early warning and precise control of orofaculum virus in sheep flocks in the southwestern plateau region. It has good prospects for promotion and significant social and economic benefits. Attached Figure Description

[0030] Figure 1 This is a sampling location map proposed in an embodiment of the present invention;

[0031] Figure 2 This is a partial PCR electrophoresis result from the Qujing area in an embodiment of the present invention;

[0032] Figure 3 This is the ORFV phylogenetic tree in the embodiments of the present invention;

[0033] Figure 4 This is a diagram showing the screening results of probe 1 primer pair in an embodiment of the present invention;

[0034] Figure 5 This is a diagram showing the screening results of probe 2 primer pair in an embodiment of the present invention;

[0035] Figure 6 This is a comparison diagram of probe primers in the embodiments of the present invention;

[0036] Figure 7 The results of gel recovery fragment amplification electrophoresis in this embodiment of the invention are shown below; where: M: DL700; N: negative control; 1-6: PCR products;

[0037] Figure 8The results of bacterial culture PCR electrophoresis in the embodiments of the present invention are shown below; where: M: DL700; N: negative control; 1-7: PCR products;

[0038] Figure 9 This is a diagram showing the ORFV plasmid sequencing results in an embodiment of the present invention;

[0039] Figure 10 This is a diagram showing the alignment results between the ORFV positive plasmid and the reference sequence in an embodiment of the present invention.

[0040] Figure 11 This is a diagram showing the specificity test results in an embodiment of the present invention;

[0041] Figure 12 This is a graph showing the sensitivity test results in an embodiment of the present invention;

[0042] Figure 13 This is a graph showing the repeatability test results in an embodiment of the present invention;

[0043] Figure 14 These are clinical sample results from embodiments of the present invention;

[0044] Figure 15 This is a bar chart comparing MIRA and PCR results in an embodiment of the present invention. Detailed Implementation

[0045] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0046] Example 1:

[0047] This embodiment uses an investigation into the ORFV infection status in some areas of Yunnan Province as an example to introduce the MIRA detection method for rapid detection of ORFV.

[0048] The cotton swabs and serum samples used in the experiment were collected from 17 farms in 8 prefectures and cities in Yunnan Province. Some samples were donated by the local agricultural bureaus. Specific sampling data are shown in Table 1, and sampling locations are listed below. Figure 1 .

[0049] Table 1: Source and Time of Cotton Swab Samples

[0050]

[0051] The virus strain used in the specificity experiments was kindly provided by the Laboratory of Veterinary Medicine, Yunnan Agricultural University, and the Kunming Customs Technology Center.

[0052] The main instruments used in this experiment are shown in Table 2.

[0053] Table 2: Information on Major Instruments

[0054]

[0055]

[0056] The main reagents used in this experiment are shown in Table 3.

[0057] Table 3: Information on Main Reagents

[0058] reagents factory LB agar medium Guangdong Huankai Microbial Technology Co., Ltd. LB broth culture medium Guangdong Huankai Microbial Technology Co., Ltd. MIRA Detection Kit (Fluorescent Type) Weifang Anpu Future Biotechnology Co., Ltd. Agarose Aikerui Biotechnology DH5α competent state Aikerui Biotechnology 50×TAE Wuhan Saiweier Biotechnology Co., Ltd. DEPC water Wuhan Saiweier Biotechnology Co., Ltd. SerRed nucleic acid dye Wuhan Saiweier Biotechnology Co., Ltd. DL2000 Beijing Quanshijin Biotechnology Co., Ltd. DL700 Tiangen Biotech (Beijing) Co., Ltd. Rapid plasmid extraction kit Tiangen Biotech (Beijing) Co., Ltd. Glue recovery kit OMEGA Biotechnology Co., Ltd. DNA / RNA Virus Extraction Kit TAKARA pMD19-T Vector Cloning Kit TAKARA 2×TaqPlusMasterMixⅡ(DyePlus) Nanjing Novozymes Biotechnology Co., Ltd. <![CDATA[ddH2O]]> Sangon Biotech (Shanghai) Co., Ltd. ampicillin solution Baisisha Bio SheepORFVELISAKit Omnimabs

[0059] The preparation of the main reagents includes:

[0060] Preparation of LB medium: Weigh 7.2g of LB agar into a glass bottle, then measure 200mL of pure water into the bottle and mix. Place the bottle in an autoclave and wait for the temperature to drop below 55℃. Add ampicillin solution (100mg / μL) at a ratio of 1μL per 1mL of LB medium. Mix thoroughly and pour into a petri dish.

[0061] Preparation of LB broth: Measure 200mL of pure water into a glass bottle using a graduated cylinder, then weigh 4.2g of LB broth powder into the bottle and mix well. Place the mixture in an autoclave and wait for the temperature to drop below 55℃ before storing it in a 4℃ refrigerator for later use.

[0062] Preparation of agarose gel: Weigh 0.6g of agarose into an Erlenmeyer flask. Measure 60mL of 1×TAE into the flask using a graduated cylinder. Gently shake the flask to mix the powder with the solution. Place the flask in a microwave oven and heat for 30 seconds. After heating, remove the flask and observe whether the powder has dissolved and whether the liquid is clear. Gently shake the flask and place it back into the microwave oven for heating. Repeat this process several times until the liquid is clear and transparent, and there are no particulate substances on the flask walls. After the solution has cooled slightly, add 6μL of SerRed nucleic acid dye. Gently shake the flask to mix the dye with the solution evenly. Pour the mixture into an electrophoresis template, remove any air bubbles, insert a comb, and wait for it to completely solidify before removing the comb.

[0063] Investigation into ORFV infection status:

[0064] 1. Sample Collection and Preservation: Blood Sample Collection and Preparation: Sheep were restrained while standing, and blood was collected from the jugular vein. Blood samples were labeled and allowed to stand at room temperature until serum precipitated. The serum was then aliquoted into centrifuge tubes, labeled, and stored at -20°C. 2. Pathogen Sample Collection: Nasal swabs were collected from the sheep. A sterile cotton swab was inserted into the sheep's nasal cavity and rotated three times, collecting one swab from each nasal cavity. After collection, the swabs were placed in cryovials, the caps were tightened, labeled, and stored in liquid nitrogen.

[0065] PCR testing:

[0066] Nucleic acid extraction: Add PBS to the collected and stored cotton swab tubes, place them on a vortex mixer and shake to mix. The specific operation method is as follows, referring to the instructions of the nucleic acid extraction kit:

[0067] Step (1) Take 200 μL of PBS suspension, add 200 μL of Buffer VGB, 20 μL of proteinase K and 1 μL of Carrier RNA, mix thoroughly and place in a 56℃ water bath for 10 min;

[0068] Step (2) Add 200 μL of anhydrous ethanol to the lysis buffer and mix thoroughly by suction and whisking.

[0069] Step (3) Transfer the solution from (2) to a centrifuge column, centrifuge at 12000 rpm for 2 min, and discard the filtrate;

[0070] Step (4) Add 500 μL of Buffer RWA to the centrifuge column, centrifuge at 12000 rpm for 2 min, and discard the filtrate;

[0071] Step (5) Add 700 μL of Buffer RWB to the centrifuge column, centrifuge at 12000 rpm for 2 min, and discard the filtrate;

[0072] Step (6) is to repeat step (5);

[0073] Step (7) Place the centrifuge column back into the collection tube and centrifuge again for 2 minutes;

[0074] Step (8) Place the centrifuge column on a new 1.5 mL centrifuge tube, add 50 μL of RNase-free H2O to the middle of the membrane, and let it stand at room temperature for 5 min;

[0075] Step (9) Centrifuge at 12000 rpm for 2 min to elute DNA;

[0076] Step (10) Detect the DNA concentration using a micro spectrophotometer, label it, and store it at -20℃.

[0077] Based on publicly available sequences from GenBank, the whole genome sequences of 11 ORFVB2L strains isolated in China and registered with NCBI were downloaded. Primers without dimers or secondary structures were selected using PrimerPremier5. The amplification primers were B2L-F: 5'-CGGCGGGCGTCAACTACTACAAG-3'; B2L-R: 5'-CGAGGTTGGCGACCGTGAAGTGC-3'. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The selected primers were used to amplify the extracted nucleic acid fragments. The PCR reaction system and conditions are shown in the table below.

[0078] Table 4: Target Fragment Amplification Reaction System

[0079] reagents Dosage FW 1.0μL RV 1.0μL 2×TaqPlusMasterMixⅡ(DyePlus) 12.5μL <![CDATA[ddH2O]]> 9.5μL DNA 1.0μL Overall system 25.0μL

[0080] Table 5: Target Fragment Amplification Reaction Procedure

[0081]

[0082] Electrophoresis: Place the pre-prepared agarose gel into the electrophoresis tank, and add DNA marker, positive control, negative control, and sample in sequence. Except for the first marker well which contains 7 μL L 1500, add 10 μL of the corresponding PCR product to each well. Adjust the electrophoresis apparatus to 400 mA, 100 V, and 40 min. After the products have migrated, transfer the gel to an imager, start the gel imaging system, and save the images for subsequent data analysis.

[0083] Product recovery and purification: After electrophoresis, cut the target fragment along the inside of the band. Follow the instructions for gel recovery kit to perform the gel recovery procedure, as follows:

[0084] Add 1 to 1.5 times the volume of BufferGDP to the centrifuge tube containing the gel block, and incubate in a water bath at 50 to 55°C for 10 to 15 minutes until the gel block is completely dissolved. Inverting the tube during the water bath can accelerate the dissolution process.

[0085] Briefly centrifuge the centrifuge tube to collect the liquid on the tube wall, place the centrifuge column inside the collection tube, transfer ≤700μL of sol solution into the centrifuge column, and centrifuge at 12000×g for 1min.

[0086] Discard the filtrate, put the centrifuge column back into the collection tube, transfer the remaining liquid completely into the column, and centrifuge at 12000×g for 1 min;

[0087] Discard the filtrate, put the column back into the collection tube, add 150 μL BufferGDP into the column, let it stand at room temperature for 1 min, and centrifuge at 12000×g for 1 min;

[0088] Discard the filtrate, add 300 μL Buffer DW2 to the column again, centrifuge at 12000×g for 2 min, open the column cap, and air dry for 5 min to completely remove ethanol;

[0089] Place the column into a 1.5 ml centrifuge tube, add 15 μL of ELution Buffer to the middle of the column membrane, let it stand at room temperature for 2 min, and then centrifuge at 12000×g for 1 min.

[0090] The DNA concentration was detected using a micro spectrophotometer, and the DNA was labeled and used promptly for subsequent experiments or stored at -20°C.

[0091] Ligation of the recovered fragment with the vector: The target fragment obtained above was ligated with the pMD-19T vector. The ligation system was as described in the instructions. The total system volume was 10 μL, containing 0.6 μL of pMD-19T vector, 4.4 μL of recovered product, and 5 μL of Solution I. Ligation was carried out at 16 °C for 3 h.

[0092] Transformation of competent cells into ligation vector: With 1 hour remaining in the above steps, turn on the ice maker. After ligation is complete, fill a glass container with ice, remove 50 μL of competent cells from the -80℃ freezer and place them on the ice. After thawing, add 5 μL of ligation product, incubate on ice for 30 minutes, then transfer to a constant temperature water bath at 42℃ for 90 seconds. After the water bath, incubate on ice again for 3 minutes. Add 1 mL of LB broth to a centrifuge tube and incubate at 37℃ with shaking until the solution becomes turbid. Take 150 μL of the bacterial culture and spread it evenly on LB medium containing 1% ampicillin. Transfer to a constant temperature incubator at 37℃ and incubate for 12 hours until single colonies grow.

[0093] Bacterial selection and PCR: Add 1 mL of LB broth containing 1% ampicillin to a 1.5 mL centrifuge tube. Pick a single colony from the culture plate and transfer it to the centrifuge tube. Incubate at 37°C with shaking until the solution becomes turbid. Perform PCR amplification on the bacterial culture according to the PCR reaction system to identify positive clones.

[0094] Genetic phylogenetic tree construction: The above steps were performed on positive samples from different locations, and the bacterial culture samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for testing. Sequence alignment was performed on NCBI based on the sequencing results. Based on the alignment results, 2-4 alignment results were selected from each location, and the positive sample sequences were aligned using Megalign software to construct a genetic phylogenetic tree.

[0095] Serological testing: First, remove the ELISA kit from the 4°C freezer and allow it to reach room temperature. Then, remove the collected and prepared serum from the -20°C freezer and thaw it at room temperature. Perform the test according to the instructions. The specific operating procedure is as follows:

[0096] Set up 2 positive wells, 2 negative wells and 1 blank control well. Do not add sample or horseradish peroxidase labeling reagent to the blank well.

[0097] Add 50 μL of positive control to the positive control well and 50 μL of negative control to the negative control well. Add 40 μL of sample diluent to the test sample well, then add 10 μL of test sample. Add the sample to the bottom of the wells of the ELISA assay plate, avoiding touching the well walls as much as possible, and mix gently.

[0098] Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes;

[0099] Prepare the washing solution: Dilute the 30x concentrated washing solution with distilled water 30 times to 600mL, and set aside.

[0100] Washing: Remove the sealing film, discard the liquid, shake dry, add washing buffer to each well, let stand for 30 seconds and then discard, repeat 5 times, and pat dry.

[0101] Add enzyme: Add 50 μL of horseradish peroxidase labeling reagent to each well, except for the blank wells;

[0102] Incubation: Same as step (3);

[0103] Washing: Same as step (5);

[0104] Color development: Add 50 μL of color developer A and 50 μL of color developer B to each well, and react at 37°C in the dark for 15 minutes;

[0105] Termination of reaction: Add 50 μL of stop solution to each well to terminate the reaction (blue turns yellow);

[0106] Detection: Zero the instrument with the blank well, and read the absorbance value at 450 nm within 15 minutes after adding the stop solution.

[0107] The data was compiled into tables to show the antibody positivity rates in different regions.

[0108] The ORFVMIRA detection method proposed in this embodiment is as follows:

[0109] Based on publicly available sequences in GenBank, the whole genome sequences of 11 ORFVB2L strains isolated in China and registered with NCBI were downloaded. After alignment of these 11 genome sequences using GeneiousPrime software, primers were designed according to the primer and probe design principles of the MIRA method. Primer lengths were designed to range from 30 to 35 bp to achieve good amplification speed and detection sensitivity; primers should not be too long or too short, with a minimum length of 25 bp. The probe length was ideally 46–52 nt. Tetrahydrofuran (THF) was used as the exonuclease marker at a position approximately 35 nt from the 5' end. The upstream T base of THF was labeled with FAM, and the downstream T base was labeled with the quencher group BHQ1. A C3-spacer was modified at the end, 15 nt from the THF. Primer and probe design was performed using PrimerPremier5; specific sequences are shown in Tables 6 and 7.

[0110] Table 6: Primer Sequence Listing for MIRA Probe 1

[0111]

[0112] Table 7: Primer Sequence Listing for MIRA Probe 2

[0113]

[0114] Viral DNA extraction: Add the collected sample to PBS and extract nucleic acid according to the steps. The extraction method is the same as the PCR detection steps.

[0115] Primer optimization in the ORFVMIRA reaction system: The two upstream primers and three downstream primers designed in Tables 6 and 7 were matched to obtain a total of 12 primer pairs. Primer matching information is shown in Tables 8 and 9. Using the MIRA detection kit (fluorescent type), primers were screened according to the reaction system (Table 10) and reaction procedure (Table 11) provided by the kit. The experimental results were compiled and recorded. The primer pair with the lowest Ct value and highest fluorescence value among the 12 primer pairs was selected for subsequent experiments.

[0116] Table 8: Primer Matching Table for MIRA Probe 1

[0117] upstream and downstream primer matching Product length (bp) F1+R1 201 F1+R2 242 F1+R3 337 F2+R1 199 F2+R2 240 F2+R3 335

[0118] Table 9: Primer Matching Table for MIRA Probe 2

[0119] upstream and downstream primer matching Product length (bp) F1+R1 205 F1+R2 266 F1+R3 210 F2+R1 337 F2+R2 398 F2+R3 342

[0120] Table 10: MIRA Reaction System

[0121]

[0122]

[0123] Table 11: MIRA Reaction Procedure Table

[0124] temperature time Cycle number 42℃ 30s 39

[0125] Optimization of the optimal reaction procedure for ORFVMIRA: While ensuring a constant reaction system, preheating at the optimal temperature before the original reaction not only promotes the reaction rate but also eliminates errors caused by improper experimental operation. Following the instructions provided with the kit, the preheating time is 3–5 min, and the reaction temperature is 39–42 °C. The time and temperature with the lowest Ct value are selected as the appropriate reaction scheme to achieve optimal reaction results.

[0126] Preparation of positive template plasmid: A positive template plasmid for ORFV is prepared to provide high-purity detection material for the subsequent sensitivity testing of the detection method. The primers selected in section 2.3.3.1 are used to amplify the target fragment. The PCR reaction system is the same as in Table 2-3, and the reaction procedure is shown in Table 12.

[0127] Table 12: PCR Reaction Procedure

[0128]

[0129] Product recovery and purification: The product obtained from the preparation of the positive template plasmid was recovered using a gel recovery kit.

[0130] Ligation of the recovered fragment with the vector: The target fragment obtained above is ligated with the pMD-19T vector, following the same steps as above.

[0131] Transforming the linker into competent cells: The linker product obtained above is transformed into competent cells, as described in the above steps.

[0132] Bacterial selection and PCR: Following the steps for constructing a phylogenetic tree, single colonies are selected, amplified and identified using the PCR system, and it is determined whether they are the target fragment.

[0133] To obtain a large quantity of bacterial culture, the bacterial culture identified as containing the target fragment during the bacterial selection and culture PCR steps was added back to LB broth containing 1% ampicillin and shaken again for 12–16 hours. After obtaining a large quantity of bacterial culture, the plasmid was extracted according to the plasmid extraction kit instructions. The specific steps are as follows:

[0134] Step (1): Centrifuge the bacterial solution at 5000×g for 10 min at room temperature and collect the bacterial cells;

[0135] Step (2): Discard the culture medium, add 500 μL of Solution / RNase A mixture to the precipitate, and vortex to completely resuspend the cells;

[0136] Step (3): Add 500 μL Solution II, gently invert and mix 8 to 10 times, and let the lysis buffer stand at room temperature for 2 to 3 minutes.

[0137] Step (4): Add 250 μL N3 Buffer, gently invert the centrifuge tube several times to mix until a white flocculent precipitate is formed, and centrifuge at 13000×g for 10 min at room temperature;

[0138] Step (5): Transfer the supernatant to a new 1.5 mL centrifuge tube, add 0.1 times the volume of the supernatant to the lysate, invert and mix 10 times, and let stand in an ice bath for 10 min.

[0139] Step (6): Place the lysis buffer from step (5) in a 42°C water bath for 5 minutes. At this point, the lysis buffer will become turbid again. Centrifuge at 12000×g for 3 minutes at 25°C. The ETRSolution will form a blue layer at the bottom of the test tube.

[0140] Step (7): Transfer the supernatant to a new 2mL centrifuge tube, add 0.5 times the volume of the supernatant in anhydrous ethanol, invert and mix 6-7 times, and let stand at room temperature for 1-2 minutes.

[0141] Step (8): Insert the centrifuge column into a 2mL collection tube, add 250μL of 3M NaOH, centrifuge at 12000×g for 1min, remove the filtrate, and activate the centrifuge column;

[0142] Step (9): Transfer ≤700μL of the mixture to a centrifuge column, centrifuge at 12000×g for 1 min at room temperature, and discard the filtrate;

[0143] Step (10), repeat step (9) until all the mixture obtained in step (7) is incorporated into the centrifuge column;

[0144] Step (11): Add 500 μL HBC Buffer to the centrifuge column, centrifuge at 12000 × g for 1 min at room temperature, and discard the filtrate;

[0145] Step (12): Add 700 μL DNAWashBuffer to the centrifuge column, centrifuge at 12000×g for 1 min at room temperature, and discard the filtrate;

[0146] Step (13): Repeat step (12) for a second wash;

[0147] Step (14): Insert the centrifuge column into the collection tube and centrifuge at 12000×g for 2 min at room temperature to dry the binding column matrix;

[0148] Step (15): Insert the centrifuge column into a clean 1.5 mL collection tube, add 100 μL LEndo-FreeElution Buffer, and let it stand at room temperature for 1 min;

[0149] Step (16): Elute DNA by centrifuging at 12000×g for 1 min;

[0150] Step (17): Detect the DNA concentration using a micro spectrophotometer, label it, and use it promptly for subsequent experiments or store it in an environment of -20℃.

[0151] Template plasmid sequencing: The extracted plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for testing, and the sequencing results were compared with the reference sequence. If the gene sequence of the extracted plasmid is consistent with the reference sequence, it indicates that the positive template plasmid has been successfully constructed and can be used for subsequent experiments.

[0152] Sensitivity experiment: Plasmids with an initial concentration of 147 ng / μL (copy number 4.00 × 10⁻⁶) were prepared during plasmid extraction. 11 Dilute with copies / μL, at a dilution of 10:-1 ~10 -11 Using positive plasmids of different dilutions as DNA templates, experiments were conducted using the MIRA detection kit (fluorescent type) under optimized conditions. Detection was performed on a real-time quantitative PCR instrument.

[0153] Specificity experiments: Nucleic acids were extracted from bluetongue virus (BTV), peste des petits ruminants virus (PPRV), pseudorabies virus (PRV), fowlpox virus (FPV), Mycoplasma gallisepticum (MG), Mycoplasma masynoviae (MS), and Mycoplasma bovis. Specificity experiments were performed using the established MIRA detection method, and Ct values ​​and fluorescence curves were recorded.

[0154] Repeatability test: Select 3 different dilutions of 10 -2 10 -4 10 -6 The ORFV positive template plasmid was used to set up three parallel samples for repeated repeatability determination, and the corresponding Ct values ​​were recorded to evaluate the repeatability of the established MIRA method.

[0155] Clinical sample testing: Sixty-two cotton swab nucleic acid samples were randomly selected from Qujing, Lijiang, and other areas in Yunnan Province. The MIRA detection method established in this experiment was used to detect ORFV, testing the method's ability to detect orthopneavirus in clinical cotton swab samples. Sample information is shown in Table 13.

[0156] Table 13 Sample Data Table

[0157] Sample source Randomly select sample number Qujing 30 Lijiang 20 Baoshan 12 total 62

[0158] Comparison of MIRA test results with PCR results

[0159] PCR test results were compared with clinical sample test results, and statistical charts were created to analyze the differences between the two methods. The positive ORFV results were statistically analyzed to determine the ORFV infection positivity rate in some areas of Yunnan Province. Specific data are shown in Table 14, and some electrophoresis results are also available. Figure 2 As shown. From the results of the phylogenetic tree ( Figure 3 It can be seen that the nine samples from the seven regions are all on independent branches.

[0160] Table 14: Statistical Table of ORFVPCR Positive Results

[0161]

[0162]

[0163] Serological results: The serum samples obtained after treatment were tested by ELISA according to the serological testing procedure. The overall ORFV antibody positivity rate of the sheep flock was 46.7%. Specific data are shown in Table 15.

[0164] Table 15: Serological Results

[0165] area Sample size Number of positive cases Positive rate Chuxiong Prefecture 43 42 97.7％ Kunming 29 10 34.5％ Nujiang Prefecture 25 15 60.0％ Lijiang 20 8 40.0％ Baoshan 141 34 24.1％ Pu'er 42 18 42.9％ Zhaotong 34 31 91.2％ Qujing 30 12 40.0％ total 364 170 46.7％

[0166] Establishment of ORFVMIRA detection method

[0167] Optimization of reaction system - primer screening

[0168] Two pairs of probes and twelve pairs of primers were designed and screened. The pair with the lowest Ct value and the highest peak value was selected as the optimal primer. The screening results are shown below. Figure 4 , Figure 5 , Figure 6 And Tables 16, 17, and 18. As can be seen from the figures, the Ct value of the upstream primer F2 and the downstream primer R3 combined with probe 1 is the smallest at 12.68, and the peak value is the highest. Moreover, the Ct value of this probe primer pair is more stable than that of probe 2. Therefore, this probe primer pair was selected as the optimal primer pair for subsequent experiments.

[0169] Table 16: Ct values ​​corresponding to the screening results of probe 1 primer pairs

[0170] primer pairs Ct value F1+R1 23.32 F1+R2 11.92 F1+R3 12.23 F2+R1 24.27 F2+R2 11.50 F2+R3 12.54 negative control -

[0171] Table 17: Ct values ​​corresponding to the screening results of probe 2 primer pairs

[0172]

[0173]

[0174] Table 18. Comparison results of probe and primer Ct values

[0175] probe primer pairs Ct value Probe 1 F2+R3 12.68 Probe 2 F2+R2 18.91 negative control -

[0176] ORFVMIRA optimal reaction system optimization: According to the kit instructions, the optimal temperature and preheating time were screened out. Preheating before the optimal reaction temperature can accelerate the reaction. The reaction conditions were optimized according to the instructions. Finally, the optimal reaction program was screened out as 42℃, preheating for 5 min (Table 19-21).

[0177] Table 19: MIRA Warm-up Time Screening Results

[0178] preheating time Ct value 3min 12.82 4min 10.7 5min 10.34

[0179] Table 20: MIRA Reaction Temperature Screening Results

[0180] reaction temperature Ct value 39℃ 10.34 40℃ 9.96 41℃ 6.97 42℃ 5.00

[0181] Table 21: MIRA Reaction Optimization Procedure Table

[0182] temperature time Cycle number 42℃ 5min 1 42℃ 30s 39

[0183] Construction of template plasmid

[0184] Target fragment amplification

[0185] The gel-recovered products were subjected to conventional PCR amplification using selected primers. The electrophoresis results of the PCR products are shown in [Figure 1]. Figure 7 This yields the target band that matches the expected fragment size.

[0186] plasmid identification

[0187] Bacterial PCR: After picking a single colony from the culture medium and shaking it, it was determined whether it was the target bacterium. The target fragment was amplified using primers selected in 2.3.3.1. The results of conventional PCR and electrophoresis confirmed that the picked colony was the target bacterium, and the amplified fragment was the same size as the target fragment (335 bp). The electrophoresis results are shown below. Figure 3-7 correspond Figure 8 .

[0188] Template plasmid sequencing: The positive plasmid was named pMD-19T-ORFV, and the sequencing results correspond to the alignment results. Figure 9-10 According to the sequencing results, the target fragment of the ORFVB2L gene has been successfully ligated into the pMD-19T vector, and the amplified ORFVB2L gene sequence has a homology of 95.8% to 99.1% with the downloaded reference sequence.

[0189] Specificity test results: Specificity tests were performed using the established method, and the results are shown below. Figure 11 ,from Figure 11 As can be seen, the curve containing ORFV nucleic acid amplified and produced a fluorescent signal, while the nucleic acids of other viruses and bacteria did not produce a fluorescent signal. This proves that the method is only effective for the detection of ORFV and has good specificity.

[0190] Sensitivity test results: The extracted plasmid was detected, and the final concentration of the plasmid was 147 ng / μL (copy number 4.00 × 10⁻⁶). 11 (copies / μL). The positive template plasmid was prepared at 10... -1 ~10 -11 The results of the serial dilution experiment, performed using MIRA technology, are as follows: Figure 3-11 correspond Figure 12As can be seen from the figure, the positive template plasmid was diluted to 10... -10 The signal was still detectable at that time, and the calculated copy number for this dilution factor was 3.24 × 10⁻⁶. 5 copies / μL.

[0191] Repeatability test results: Three different dilutions of 10 were randomly selected. -2 10 -4 10 -6 (The copy number is 5.89 × 10) 9 copies / μL, 2.80×10 9 copies / μL, 2.07×10 9 The positive template plasmid (copies / μL) was used for repeatability experiments. Three parallel samples were set up for each dilution, and the results are shown in [Figure number missing]. Figure 13 Table 22 shows that the fluorescence curves of samples at the same dilution exhibit good repeatability.

[0192] Table 22: Ct values ​​corresponding to repeatability experiment results

[0193]

[0194] Clinical sample testing results: A total of 62 cotton swab samples were randomly selected from Qujing, Lijiang, and other areas, and nucleic acid testing was performed using the MIRA detection method established in this experiment. The results showed that 42 out of the 62 samples were positive, with a detection rate of 67.7%. The detection rate was 83.3% in Qujing, 75% in Lijiang, and 40% in Baoshan, with positive samples detected in each area. This indicates that the ORFVMIRA detection method established in this experiment can effectively detect orthopneavirus in cotton swab nucleic acid. Specific data are shown in Table 23. Figure 14 .

[0195] Table 23: Clinical Sample Test Results

[0196]

[0197]

[0198] Comparison of MIRA results and PCR results

[0199] A statistical chart was created based on the serological results in 3.1.2 and the clinical sample test results in 3.2.6. Figure 15 ),from Figure 15As can be seen, the detection rate of MIRA in randomly selected clinical samples was much higher than that of PCR. In Qujing, the detection rate of MIRA in 30 samples was 83.3%, while that of PCR was 6.7%; in Lijiang, the detection rate of MIRA in 20 samples was 75%, while that of PCR was 30%; and in Baoshan, the detection rate of MIRA in 12 samples was 40%, while that of PCR was 0%.

[0200] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rapid detection method for sheep poxvirus based on MIRA technology, characterized in that, The method includes the following steps: Step 1: Extract nucleic acid from the sample to be tested; Step 2: Using the extracted nucleic acid as a template, perform isothermal amplification using the MIRA reaction system; Step 3, wherein the MIRA reaction system comprises: The specific primer pair targeting the ORFVB2L gene has the sequences shown in SEQ ID NO:1 and SEQ ID NO:2; Fluorescent probe, sequence as shown in SEQ ID NO:3; And MIRA basic reaction reagents; Step 4: React at a constant temperature of 42℃ for 5-20 minutes, and judge the results by real-time fluorescence detection.

2. The rapid detection method for sheep poxvirus based on MIRA technology according to claim 1, wherein, The MIRA reaction system was preheated for 5 minutes before the reaction.

3. The rapid detection method for sheep poxvirus based on MIRA technology according to claim 1 or 2, wherein, The probe is a FAM / BHQ1 dual-labeled probe, with a tetrahydrofuran cleavage site at the 35th nucleotide position at its 5' end.

4. The rapid detection method for sheep poxvirus based on MIRA technology according to any one of claims 1-3, wherein, The detection limit of the method is 7.63 × 10⁻⁶. 9 copies / μL.

5. The rapid detection method for sheep poxvirus based on MIRA technology according to any one of claims 1-4, wherein, The samples to be tested are sheep nasal swabs, serum, or diseased tissue.

6. A MIRA kit for detecting sheep poxvirus, characterized in that, The kit contains: The specific primer pair SEQ ID NO:1 and SEQ ID NO:2 as described in claim 1; The fluorescent probe SEQ ID NO:3 as described in claim 1; The basic reaction reagents for MIRA include recombinase, DNA polymerase, single-stranded binding protein, dNTPs, buffer, and magnesium ions.

7. The kit according to claim 6, wherein, The kit is suitable for isothermal reactions at 42°C and does not require a thermal cycler.

8. The application of the method according to any one of claims 1-5 or the kit according to claims 6-7 in the rapid on-site diagnosis of poxvirus.

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