Primer group for detecting main pathogen CVA24v of acute hemorrhagic conjunctivitis based on VP1 region and application of primer group

Abstract

The invention relates to the technical field of gene engineering, in particular to a primer group for detecting a main pathogen CVA24v of acute hemorrhagic conjunctivitis based on a VP1 region and application of the primer group. The invention provides a primer group for detecting a main pathogen CVA24v of acute hemorrhagic conjunctivitis. The primer group comprises a primer pair and a probe, the nucleotide sequences of the primer pair are as shown in SEQ ID NO.1 and SEQ ID NO.3; the nucleotide sequence of the probe is as shown in SEQ ID NO. 2. The method has reliable repeatability and consistency in practical application, and is suitable for rapid and accurate identification of the main pathogen CVA24v of acute hemorrhagic conjunctivitis in clinical samples and large-scale epidemiological monitoring.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a primer set for detecting CVA24v, the main pathogen of acute hemorrhagic conjunctivitis, based on the VP1 region, and its application. Background Technology

[0002] Acute hemorrhagic conjunctivitis (AHC) is characterized by severe conjunctival hyperemia, often accompanied by a strong foreign body sensation, tearing, photophobia, and increased discharge. AHC has garnered widespread attention in global public health due to its short incubation period, rapid onset, and rapid spread; its incidence rate is second only to influenza and hand-foot-and-mouth disease.

[0003] Many viruses can cause enterovirus infection (AHC). However, the main pathogen causing global AHC outbreaks is currently Coxsackievirus A24 variant (CVA24v). CVA24v is a human enterovirus that is transmitted through contact, such as through contact with items contaminated with the eye secretions of infected individuals. CVA24v is the most important pathogen causing AHC outbreaks both globally and domestically. Therefore, strengthening the detection of CVA24v is of great significance for effectively controlling the spread of AHC.

[0004] Currently reported primers for CVA24v quantitative PCR detection are designed based on the 5'UTR region of CVA24v, using sequences from 13 foreign strains (from Congo, French Guiana, and Guadeloupe) prior to 2007. However, since 2007, several global outbreaks of AHC caused by CVA24v have occurred. These frequent outbreaks have led to continuous mutations in the CVA24v gene, causing previously designed primers and probes to become incompatible. Furthermore, these previously designed primers and probes, based on foreign sequences, are not well-suited for detecting the currently prevalent CVA24v strains in China. More importantly, recent studies have shown frequent recombination events in the 5'UTR region of CVA24v, which alters the viral sequence. Using the 5'UTR region as the target gene for detection can lead to erroneous results. To avoid this, we focused our primer and probe design on the VP1 region. The VP1 region is the key region where the CVA24v antigenic determinant is located. This region is highly specific and relatively conserved, and the probability of recombination events is extremely low. It is widely used for the detection and genotyping of other viruses that belong to the same human enterovirus family as CVA24v. Summary of the Invention

[0005] Based on the current status of CVA24v quantitative fluorescence detection, and to avoid erroneous detection results caused by recombination events in the 5'UTR region, this invention designs a CVA24v detection probe and primers based on the VP1 region. Furthermore, the probe and primers designed in this invention are compatible with the currently prevalent CVA24v in China.

[0006] This invention, based on the VP1 region of the virus, designs a pair of specific primers targeting the currently prevalent CVA24v, enabling rapid and accurate detection of CVA24v. Using "coxsackievirus A24" and "enterovirus A24" as index terms (data collection up to October 1, 2024), this invention collected all full-length VP1 sequences of CVA24v from the GenBank database. To maximize the results of subsequent analysis, the downloaded sequences were screened. Sequences were filtered based on sequence information, removing duplicate experimental strains, cloned strains, and strains with unclear date regions, etc. Then, MAFFT was used for multiple sequence alignment to remove sequences containing large deletions, ORF regions containing "N" bases, and non-CVA24v sequences. Finally, a dataset of 257 qualified VP1 sequences was selected. This dataset contains VP1 sequences from 1988 to 2023, covering strain sequences from multiple AHC outbreaks both domestically and internationally. This dataset is comprehensive, and probes designed based on it are reliable. The final designed probe primer sequences are shown in SEQ ID NO.1~SEQ ID NO.3.

[0007] SEQ ID NO.1: CVA24v-YF: CCA GAT TYG ACC TAG ARA TGA C

[0008] SEQ ID NO.2: CVA24v-DP:TCA TCG AAC CCG TCA GTR TT

[0009] SEQ ID NO.3: CVA24v-MR: CAA CRG TCT CAT CYT TCA AAG G

[0010] (Explanation of degenerate base codes: Y=C / T; R=A / G)

[0011] Preferably, the gene number of the CVA24v gene is NMDCN0006499.

[0012] The present invention also provides the application of the primer set described above in the preparation of a detection kit for CVA24v, the main pathogen of acute hemorrhagic conjunctivitis.

[0013] Preferably, the detection method corresponding to the kit is: (1) Obtain conjunctival swabs or viral isolates from the patient as samples; (2) The sample was amplified by fluorescence quantitative PCR using the primer set described above, and the amplification results were determined.

[0014] Preferably, the amplification system is shown in Table 1: Table 1 Amplification System

[0015] Preferably, the amplification procedure is shown in Table 2: Table 2 Amplification Procedure

[0016] Preferably, the concentrations of the forward primer, probe, and reverse primer are all 15~25 µmol / L.

[0017] Preferably, the criteria for determining the amplification result are: a Ct value greater than 35 is negative, and a Ct value less than or equal to 35 is positive.

[0018] The present invention also provides a detection kit for CVA24v, the main pathogen of acute hemorrhagic conjunctivitis, including the primer set described above.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The fluorescent quantitative detection primers and probes for the VP1 coding region of CVA24v, the main pathogen of acute hemorrhagic conjunctivitis, provided by this invention, exhibit excellent technical performance in terms of sensitivity, specificity, and repeatability. Experimental results show that the limit of detection (LOD) of this method can reach 10² copies per microliter, and only 12 copies are needed per reaction to achieve stable detection. The coefficient of determination (R²) of the standard curve is higher than 0.99, demonstrating good quantitative ability and detection sensitivity.

[0020] In terms of specificity, the primer probe can accurately distinguish CVA24v from 74 other human enteroviruses without cross-reactivity, indicating that it has high type specificity and effectively avoids detection errors caused by potential recombination events in the 5'UTR region.

[0021] Furthermore, the detection method exhibited extremely high stability in repeatability experiments. The results of three repeated tests for five concentration gradients showed that the standard deviation (SD) was less than 0.5 and the coefficient of variation (CV) was less than 1.2%, indicating that the method has reliable repeatability and consistency in practical applications and is suitable for rapid and accurate identification of CVA24v in clinical samples and large-scale epidemiological surveillance. Attached Figure Description

[0022] Figure 1 Specificity evaluation of the CVA24v probe.

[0023] Figure 2 Detect the dynamic range and linear equation for CVA24v.

[0024] Figure 3 Evaluation of the sensitivity of the CVA24v probe.

[0025] Figure 4 Specific bands were amplified in all 42 fluorescent positive samples. Detailed Implementation

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1

[0028] 1. Validation of CVA24v VP1 region probe primers

[0029] This invention primarily validates the designed probe in terms of sensitivity, specificity, and stability to demonstrate the effectiveness of the technology. A CVA24v standard was constructed using plasmids, and this standard was used to test the designed fluorescent probe. CVA24v contains four genotypes, which show high similarity. We used the GⅣ genotype isolated in 2023 (gene number NMDCN0006499, National Microbial Science Data Center (NMDC)). VP1 The target sequence was constructed into the pUC57 vector.

[0030] The synthesized plasmid was then transformed, and the plasmid powder containing the target sequence was centrifuged at 5000 rpm for 1 minute. After centrifugation, 20 μL of enzyme-free water was added to the bottom of the test tube. 20 μL of competent cells were transferred to a 1.5 mL sterile EP tube, followed by 2 μL of diluted plasmid. The tube was then heat-shocked at 42°C for 90 seconds, followed by 5 minutes on ice. 500 μL of SOC medium was added to the tube, and the tube was shaken at 37°C, 220 rpm for 30 minutes. After shaking, 100 μL was spread onto ampicillin-resistant LB agar and incubated upside down at 37°C for 12 hours. After incubation, the culture was removed and temporarily stored at 4°C (too long a time will cause bacteria to form satellite colonies), and a bacterial suspension (1:9) was prepared.

[0031] Pick bacteria from the plate and transfer them to a 15mL centrifuge tube filled with liquid. Loosen the cap to allow air contact and incubate at 37°C for 12 hours. Perform a small-scale extraction of the transformed plasmid by centrifuging the shaken *E. coli* at 4,200 rpm for 20 minutes. Discard the liquid and invert the tube onto absorbent paper to drain. Remove the adsorption column C. P3 Place the column in a collection tube, add 500 μL of equilibration buffer BL, cap, and centrifuge at 12,000 rpm for 1 min. Discard the waste liquid in the collection tube, retain the adsorption column, and return it to the collection tube. Take 5 mL of overnight cultured E. coli and add it to the centrifuge tube. Centrifuge at 12,000 rpm for 1 min and discard the supernatant. Add 250 μL of solution P1 to the centrifuge tube containing the precipitate and resuspend the bacterial precipitate using a pipette or vortex mixer. Add 250 μL of solution P2 to the centrifuge tube and gently invert 6 times to fully lyse the bacteria, being careful not to damage the genomic DNA. The process should not exceed 5 min. Add 350 μL of solution P3 to the centrifuge tube and quickly and gently invert 8 times. A white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 min, collect the supernatant, and centrifuge again at 12,000 rpm for 30 sec. Discard the waste liquid in the collection tube and place the adsorption column C... P3 Continue adding to the collection tube. Add 600 μL of wash buffer PW containing anhydrous ethanol to the adsorption column CP3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid in the collection tube, and continue the adsorption column C... P3 Continue placing it into the collection tube and repeat the above steps once.

[0032] To remove residual wash solution from the adsorption column, centrifuge at 12,000 rpm for 2 min. Finally, place the adsorption column CP3 into a new centrifuge tube, add 50 μL of elution buffer EB to the middle region of the adsorption membrane, incubate at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min, and collect the plasmid.

[0033] Next, in vitro transcription was performed using RiboMAX. TMThe Large Scale RNA Production System-T7 kit was used for in vitro transcription of purified linear plasmids. The reaction system was constructed as follows: 5 μL T7 Transcription 5X Buffer, 7.5 μL rNTP mixture (25 mM ATP, CTP, GTP, UTP), 2 μg linearized DNA template, 2.5 μL enzyme mixture (T7), and 8 μL nuclease-free water, for a total reaction volume of 25 μL. The mixture was vortexed to ensure thorough mixing and then incubated at 37°C for 4 h. Next, the standards were purified by adding 200 μL of enzyme-free water to the transcribed product and mixing. Then, 200 μL of lysis buffer (prepared by adding 10 μL β-mercaptoethanol to 1 mL of lysis buffer) and 200 μL of anhydrous ethanol were added, and the mixture was thoroughly mixed and allowed to stand for 5 min. After settling, transfer the sample to a column and allow it to adsorb for 2 minutes. Centrifuge at 12,000 rpm for 1 minute, add 700 μL Wash Buffer I, and centrifuge at 12,000 rpm for 1 minute. Add 500 μL Wash Buffer II, and centrifuge at 12,000 rpm for 1 minute. Repeat this step. Centrifuge at 12,000 rpm for 1 minute (empty space), transfer to a new collection tube, add 40 μL of Nuclease-free water for elution, and perform one more centrifugation to complete the elution process. Use Qubit. TM The RNABRAssay kit is used to quantify purified RNA, calculate RNA copy number, and dilute standards for subsequent probe evaluation.

[0034] 1.1 Probe sensitivity

[0035] The CVA24v standard was precisely analyzed spectrophotometrically using the Qubit™ RNA BR Assay kit to calculate its copy number. The formula for calculating the copy number is as follows:

[0036] Note: NA represents the number of molecules per mole, and Avogadro's constant (6.02 × 10⁻⁶). 23 ); concentration is the RNA concentration measured by a spectrophotometer; transcript length is the length of the standard bases.

[0037] Subsequently, the standard was serially diluted 10-fold using Nuclease-free H2O to prepare a solution covering 10... 8Eight dilutions were prepared up to 10¹ copy number. Real-time RT-PCR experiments were performed on the standards at these eight different dilution gradients, and the threshold cycle (Ct) for each gradient was recorded in detail. The quantitative PCR system and procedure are shown in Tables 3 and 4.

[0038] Table 3. Reaction system for CVA24v Real-time RT-PCR

[0039] Table 4 Reaction conditions for CVA24v Real-time RT-PCR

[0040] Regarding result interpretation, a Ct value greater than 35 is considered a negative result, while a Ct value less than or equal to 35 is considered a positive result. This criterion allows for precise determination of the limit of detection (LOD) of the primer and probe, as well as the plotting of a standard curve and the calculation of the relevant coefficient of determination (R²). 2 ), R 2 The value can be used to evaluate the quantitative range of primers and probes, enabling more precise quantitative analysis.

[0041] The CVA24v standard was serially diluted 10-fold with enzyme-free water to obtain 10 different concentration gradients of the standard (10). 10 ~10¹ copies / μL). Subsequently, these gradient standards were subjected to real-time RT-PCR experiments, and the corresponding Ct values ​​were recorded. A standard curve was plotted with the logarithm of the copy number (Log value) on the x-axis and the Ct value on the y-axis, as shown below. Figure 1 As shown. Based on this standard curve, a line graph was plotted to calculate R. 2 All values ​​are above 0.99, and the LOD is 10. 2 Copy number / μL, 12 copies per reaction, e.g. Figure 2 As shown in the figure. These results demonstrate that the proposed detection method possesses good sensitivity.

[0042] 1.2 Probe Specificity

[0043] CVA24v standard and other human enteroviruses were simultaneously identified by real-time quantitative PCR. Seventy-four human enterovirus strains were selected from our laboratory's strain library, including 16 serotypes of human enterovirus group A (CV-A2~A8, CVA10, CV-A12, CV-A14, CV-A16, EV-A71, EV-A76, EV-A89, EV-A90, EVA120). Group B 45 types of CV-B1~B6, CV-A9, E-1~7, E-9, E-11~21, E-24~27, E-29~33, EVB74~B75, EV-B79~B81, EV-B83, EV-B85, EV-B93, EV-B97, EVB106; Group C contains 11 species: PV-1, PV-3, CV-A1, CV-A11, CV-A13, CV-A17, CV-A20, CV-A21, CVA24, EV-C96, and EV-C99. Group D has two types (EV-D68, EV-D70); Nucleic acid was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) according to the kit's instructions.

[0044] In this experiment, following the above detection method, 74 serotypes of human enteroviruses were detected together with the CVA24v standard using real-time RT-PCR. The amplification curve results showed that the established real-time RT-PCR detection method could only specifically detect CVA24v and could not detect other enteroviruses. Figure 3 This result fully confirms that the detection method has good specificity.

[0045] 1.3 Probe repeatability

[0046] Using Qubit TM The RNA BR Assay kit was used to quantify the standards, calculating the corresponding copy number using a formula. Subsequently, the standards were serially diluted 10-fold with nuclease-free water to prepare concentrations ranging from 10⁻⁶ to 10⁻⁶. 7 ~10 3 Copy number / μL dilution buffer. For each standard, three parallel real-time RT-PCR experiments were performed at five different dilution gradients, and the Ct values ​​for each group were recorded in detail. Then, the standard deviation (SD) and coefficient of variation (CV) of the Ct values ​​were calculated for each gradient's three parallel experiments to assess the stability of the primers and probes in real-time RT-PCR experiments. SD is a key indicator of the variability of Ct values; a higher SD value indicates greater variability. CV (calculated as standard deviation divided by the mean) reflects the magnitude of relative variation; a higher CV value indicates greater relative variation.

[0047] CVA24v standard was serially diluted 10-fold with enzyme-free water to prepare concentrations ranging from 1... Five dilution gradients of standards up to 10³ copies / μL were used. For these five CVA24v standards, three replicate experiments were performed, and the Ct values ​​for each experiment were recorded. Statistical analysis showed that the standard deviation (SD) of each of the three replicates was less than 0.5, and the coefficient of variation (Ct) was... CV All values ​​were less than 1.2%, and detailed results are shown in Table 5.

[0048] Table 5. Intergroup repeatability of CVA24v real-time RT-PCR

[0049] Example 2: Real-time RT-PCR validation of virus isolates from previous outbreaks

[0050] Real-time RT-PCR testing was performed on 71 virus isolates identified as CVA24v from multiple provinces in the years when AHC outbreaks occurred nationwide (1988, 1994, 2007, 2010, and 2023). The virus isolates came from the following sources: Beijing (9 isolates in 1988, 7 isolates in 1994, and 9 isolates in 2007), Hubei (11 isolates in 2010), Jiangxi (20 isolates in 2010), Yunnan (11 isolates in 2010), and Guangdong (6 isolates in 2010).

[0051] 1. Experimental materials

[0052] The fluorescent probes and primer sequences used for real-time RT-PCR are shown in SEQ ID NO.1 to SEQ ID NO.3, the RNA extraction kit RNeasy Mini kit (QIAGEN, Germany), and the PCR reaction kit (SuperScript® Ⅲ Real-time RT-PCR with Platinum® Taq)

[0053] 2. Instructions for use

[0054] 2.1 First step: Nucleic acid extraction

[0055] Nucleic acid extraction was performed on viral isolates identified as CVA24v from outbreaks in 1998, 1994, 2007, and 2010 across the country. The nucleic acid extraction steps are as follows: (1) When using the kit for the first time, the reagents need to be prepared: dilute the AW1 and AW2 buffers with anhydrous ethanol according to the volume indicated on the reagent bottle; add 310 μl of AVE buffer to the bottle containing 310 μg of Carrier RNA dry powder, vortex to mix, and centrifuge briefly. Add 310 μL of the above mixture to AVL (31 mL) to prepare AVL working solution, mix well and set aside. After all reagents are prepared, label the bottle caps and indicate the preparation date; (2) Take a 1.5 mL centrifuge tube, add 560 μl of AVL working solution and 140 μL of sample, shake to mix for 15 s, and let stand at room temperature for 10 min; (3) After instantaneous centrifugation, add 560 μl of anhydrous ethanol, shake to mix for 15 s, and then centrifuge again. (4) Pipette 630 μL of the mixture into the separation column, centrifuge at 8000 rpm for 1 min, and replace the collection tube; (5) Repeat step (4); (6) Add 500 μl of AW1 buffer to the separation column, centrifuge at 8000 rpm for 1 min, and replace the collection tube; (7) Add 500 μl of AW2 buffer to the separation column, centrifuge at 14000 rpm for 3 min, and replace the collection tube; centrifuge again at 14000 rpm for 1 min. (8) Place the separation column into a new 1.5 mL centrifuge tube, add 50 μL AVEBuffer, place at room temperature for 1 minute, then centrifuge at 8000 rpm for 1 min, discard the separation column, obtain the total viral RNA, place it in a 4℃ refrigerator or on ice for later use, or place it in a -70℃ refrigerator for long-term storage.

[0056] 2.2 Real-time RT-PCR

[0057] The extracted nucleic acid was subjected to real-time RT-PCR according to the reaction system and conditions described above. A positive control (nucleic acid from strains identified as enteroviruses and adenoviruses used as RNA templates) and a negative control (enzyme-free water instead of RNA templates) were added. A CT value showing "Undetermined" and no peak in the fluorescence curve indicates a negative result; a CT value <35.0 and a distinct "S"-shaped fluorescence curve indicate a positive result; if a specific CT value is available but the fluorescence curve shows no obvious peak, the experiment needs to be repeated for verification. If the repeated experiment shows "Undetermined," the result is considered negative; otherwise, it is considered positive.

[0058] 3. Experimental Results

[0059] The results showed that the CVA24v primers and probes designed in this experiment were used to detect 71 viral isolates identified as CVA24v in the outbreaks in 1998, 1994, 2007, and 2010 across the country, with a positive concordance rate of 100%. This indicates that the fluorescent probes and primers of this invention have good detection performance.

[0060] Example 3: Detection of conjunctival swabs collected during the 2023 AHC outbreak

[0061] Based on existing reports, we collected conjunctival swabs from multiple AHC outbreak areas in 2023 and used the primers and probes developed in this study to detect CVA24v in the swabs. The conjunctival swabs from 2023 came from the following sources: Fujian (46 samples), Hainan (22 samples), Jiangxi (4 samples), and Yunnan (6 samples).

[0062] 1. Experimental materials

[0063] The fluorescent probes and primer sequences used for real-time RT-PCR are shown in SEQ ID NO.1 to SEQ ID NO.3, the RNA extraction kit RNeasy Mini kit (QIAGEN, Germany), and the PCR reaction kit (SuperScript® Ⅲ Real-time RT-PCR with Platinum® Taq)

[0064] 2. Instructions for use

[0065] 2.1 Processing of conjunctival swab specimens

[0066] (1) Stir the received swabs thoroughly in the preservation solution at least 40 times to ensure that pathogens (such as viruses, bacteria, etc.) attached to the swabs and cells containing pathogens are fully washed away. When stirring, be careful to use moderate force to avoid damaging the swabs or container; (2) Centrifuge the swab containing the swab eluent at 10,000 rpm for 20 minutes at 4°C. This will precipitate larger cell fragments and impurities, allowing pathogens to accumulate in the supernatant; (3) After centrifugation, the supernatant was collected into a new sterile tube for subsequent experiments.

[0067] 2.2 Nucleic Acid Extraction

[0068] Nucleic acid extraction was performed on the centrifuged conjunctival swabs following the steps in Example 1.

[0069] 2.3 Real-time RT-PCR

[0070] The extracted nucleic acid was subjected to real-time RT-PCR according to the reaction system and conditions of Example 1 above, with a negative control (enzyme-free water) added at the same time. The judgment criteria were the same as in Example 1, and the fluorescent positive samples were recorded.

[0071] 2.4 Target gene amplification, electrophoresis, and sequencing verification

[0072] (1) Take 5 μL of fluorescently positive RNA sample (Ct≤35) and transfer it to a sterile 200 μL PCR tube without enzymes (in this example, VP1 region sequencing and genotyping are performed on all positive samples). (2) Prepare the PCR Mix according to the system in Table 6 (this step uses the standard RT-PCR system SuperScript®ⅢOne-Step RT-PCR with Platinum® Taq): Table 6 PCR Reaction System

[0073] (3) Perform DNA level electrophoresis on 1.5% agarose gel electrophoresis on the PCR product reaction product, cut the gel of the band and perform gel recovery experiment. For details, please refer to StartPrep rapid DNA gel recovery kit. (4) The obtained gel recovery product was sent to the company for sequencing. The sequencing yielded the complete genome sequence of the VP1 region of the enterovirus to be tested. The sequencing results were compared with the VP1 sequence of the CVA24v prototype strain using Sequencher 5.4.5 software (GeneCode, Ann Arbor, Michigan, USA). The sequences with poor quality were carefully observed and corrected. Finally, the complete VP1 region sequence was obtained. The VP1 sequence was then compared with the VP1 sequence of the CVA24v prototype strain to obtain the genotyping results of the VP1 sequence of the CVA24v prototype strain to be tested.

[0074] 3. Experimental Results

[0075] Of the 78 new conjunctival swabs collected in 2023, 42 were positive, a positivity rate of 53.85%. All 42 fluorescently positive samples amplified specific bands, as shown in the agarose gel images. Figure 4 As shown.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer set for detecting an acute hemorrhagic conjunctivitis pathogen, characterized by, The primer set comprises a primer pair and a probe; the nucleotide sequence of the primer pair is shown as SEQ ID NO. 1 and 3; and the nucleotide sequence of the probe is shown as SEQ ID NO.

2.

2. The primer set according to claim 1, characterized in that, The primer set is used for amplifying the CVA24v gene VP1 region of the conjunctivitis pathogen.

3. The primer set of claim 2, wherein, The gene number of the CVA24v gene is NMDCN0006499.

4. Use of the primer set in any of claims 1-3 in the preparation of a pathogen detection kit for acute hemorrhagic conjunctivitis.

5. Use according to claim 4, characterized in that, The detection method corresponding to the kit is: (1) extracting a swab or virus of a patient as a sample; (2) performing fluorescent quantitative PCR amplification on the sample by using the primer set in any of claims 1-3, and determining the amplification result.

6. Use according to claim 5, characterized in that, The amplification system is shown in Table 1: Table 1 Amplification system 。 7. Use according to claim 5, characterized in that, The amplification procedure is shown in Table 2: Table 2 Amplification procedure 。 8. Use according to claim 6, characterized in that, The concentrations of the forward primer, the probe, and the reverse primer are all 15-25 µmol / L.

9. Use according to claim 5, characterized in that, The determination standard of the amplification result is that a Ct value greater than 35 is negative, and a Ct value less than or equal to 35 is positive.

10. A diagnostic kit for acute hemorrhagic conjunctivitis pathogen, characterized by, The primer set in any of claims 1-3 is included.

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