Primer probe combination, freeze-drying kit and method for detecting EV71, CA16 and universal enteroviruses

Abstract

The invention discloses a primer probe combination for detecting EV71, CA16 and universal enteroviruses. The sequences in the primer probe combination are shown as SEQ ID NO.1-SEQ ID NO.17. The primer probe combination is used for targeted amplification and detection of the universal enteroviruses, the EV71, the CA16 and a human-derived interior label. A working solution of the primer probe combination and a PCR detection reagent are mixed according to the proportion of 1: 2, and a PCR premixed solution is obtained; the freeze-dried reagent can be directly added into nucleic acid of a sample to be detected, and PCR (Polymerase Chain Reaction) detection is carried out. The kit is simple and convenient to operate, can be stored and transported at room temperature, can control the detection time to be about 40 minutes, improves the detection convenience and efficiency, and has good specificity and sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a primer-probe combination, a freeze-dried kit and a method for detecting EV71, CA16 and universal enterovirus. Background Art

[0002] Enterovirus belongs to the genus Enterovirus of the Picornaviridae family. There are many types of enterovirus that can cause human disease, including four genotypes: A, B, C, and D. According to the serotype classification, they are mainly: poliovirus, Coxsackievirus A (CA), Coxsackievirus B (CB), Echovirus, new enterovirus, etc. New enteroviruses were isolated after 1969, such as the new enterovirus 71 (EV71). Enterovirus mainly infects infants and young children, mostly occurring in children under 5 years old. The average incubation period is 3-7 days. It has obvious seasonality, with the peak of epidemic in summer. The main transmission route is fecal-oral route, and it can also be transmitted through the respiratory tract. It can cause respiratory tract infection, herpetic pharyngitis, hand, foot and mouth disease, epidemic chest pain, myocarditis and pericarditis, eye disease and aseptic meningitis and other diseases. It is highly contagious and has a high latent infection rate, which can easily lead to disease outbreaks and bring serious public health problems. Enteroviruses that cause hand, foot and mouth disease mainly include: Coxsackievirus A group 4-7, 9, 10, 16 and B group 1-3, 5, some serotypes of echovirus and enterovirus 71, among which CA16 and EA71 are the most common. EV71 can cause neurological symptoms such as aseptic meningitis, brainstem encephalitis, encephalomyelitis, neurogenic myocarditis, acute flaccid paralysis, neurogenic pulmonary edema, etc., which can be life-threatening in severe cases. Therefore, timely and accurate diagnosis is crucial for the prevention and treatment of enterovirus infection.

[0003] Traditional enterovirus detection methods mainly include virus isolation and culture and serological diagnosis, but there are problems such as cumbersome operation and low sensitivity, which are prone to missed detection and cannot meet the requirements of early diagnosis. Compared with traditional detection methods, nucleic acid detection has higher sensitivity and specificity, which is helpful for early diagnosis and timely treatment in clinical practice, and has gradually become the mainstream method for enterovirus detection. At present, the nucleic acid detection technologies used for enterovirus detection mainly include real-time fluorescence quantitative RT-PCR, isothermal amplification technology, gene chip method, etc. Real-time fluorescence quantitative RT-PCR technology has the characteristics of simple operation, fast speed, low cost, high sensitivity, good specificity, etc., and is widely used in clinical practice. However, most of the enterovirus real-time fluorescence quantitative RT-PCR detection kits currently used in clinical practice are liquid reagents, which require fresh PCR reaction solution when used, and need to be frozen and transported by cold chain; they can cover fewer enterovirus types, and EV71 and CA16 are not typed and detected at the same time, and the detection time is mostly more than 1 hour; there are defects in detection efficiency and specificity, reagent storage and transportation, etc. Summary of the invention

[0004] In order to solve at least one of the above problems, the present invention provides a primer probe combination, a freeze-dried kit and a method for detecting EV71, CA16 and universal enterovirus; EV71, CA16 and universal enterovirus can be detected simultaneously, the reagent is in a freeze-dried form, and when used, there is no need to prepare a PCR reaction solution, and nucleic acid can be directly added, which is easy to operate.

[0005] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a primer-probe combination for detecting EV71, CA16 and universal enterovirus, comprising: (1) a primer pair for targeted amplification of universal enterovirus as shown in SEQ ID NO.1-SEQ ID NO.2; a probe for detecting universal enterovirus as shown in SEQ ID NO.3; the universal enterovirus includes four genotypes: A, B, C, and D; (2) The primer pairs for targeted amplification of EV71 are shown in SEQ ID NO.4-SEQ ID NO.5, SEQ ID NO.7-SEQ ID NO.8; the probes for detecting EV71 are shown in SEQ ID NO.6 and SEQ ID NO.9; (3) The primer pairs for targeted amplification of CA16 are shown as SEQ ID NO.10-SEQ ID NO.11 and SEQ ID NO.12-SEQID NO.13; the probe for detecting CA16 is shown as SEQ ID NO.14.

[0006] In some embodiments of the present invention, further, it also includes: a primer pair for targeted amplification of a human internal standard as shown in SEQ ID NO.15-SEQ ID NO.16; and a probe for detecting a human internal standard as shown in SEQ ID NO.17.

[0007] In some embodiments of the present invention, in the primer-probe combination, the 5' end of the probe is provided with a fluorescent group, the 3' end is provided with a quenching group, the fluorescent groups on the probes detecting the same target are the same, and the fluorescent groups on the probes detecting different targets are different; In some embodiments of the present invention, the fluorescent group is one of FAM, HEX, VIC, ROX and CY5, and the quenching group is one of BHQ1 and BHQ2.

[0008] In some embodiments of the present invention, the 5' end of the universal enterovirus probe in the primer probe combination is modified with a FAM fluorescent group, and the 3' end is modified with a BHQ1 quenching group; the 5' end of the EV71 probe is modified with a HEX fluorescent group, and the 3' end is modified with a BHQ1 quenching group; the 5' end of the CA16 probe is modified with a ROX fluorescent group, and the 3' end is modified with a BHQ2 quenching group; the 5' end of the human internal standard probe is modified with a CY5 fluorescent group, and the 3' end is modified with a BHQ2 quenching group.

[0009] The second aspect of the present invention provides a method for preparing a lyophilized reagent for detecting EV71, CA16 and universal enterovirus, comprising the following steps: (1) Prepare primer-probe working solution: The primer-probe dry powders in the primer-probe combination for detecting EV71, CA16 and universal enterovirus were first diluted with TE Buffer, and then the primer-probe working solution was prepared: the final concentration of the primer pair for amplifying universal enterovirus was 1 μM, and the final concentration of the probe was 0.4 μM; the final concentration of the primer pair for amplifying EV71 was 0.8 μM, and the final concentration of the probe was 0.4 μM; the final concentration of the primer pair for amplifying CA16 was 0.4 μM, and the final concentration of the probe was 0.2 μM; the final concentration of the primer pair for amplifying the human internal standard was 0.4 μM, and the final concentration of the probe was 0.3 μM; (2) Prepare PCR premix: Mix the primer probe working solution and PCR detection reagent in a ratio of 1:2 to obtain a PCR premix; (3) Freeze-drying procedure: The PCR premix is ​​divided into aliquots and freeze-dried using the following freeze-drying procedure to obtain the freeze-dried reagent: A. Pre-freezing: plate temperature -45°C, cooling time 60min, holding time 120min; B. Sublimation drying: board temperature -25℃, heating time 60min, hold time 600min, pressure 10Pa; C. Analytical drying: plate temperature 30°C, heating time 120min, hold time 120min, pressure 10Pa.

[0010] In some preferred embodiments of the present invention, the PCR detection reagent in step (2) is a self-prepared PCR detection reagent or a commercial PCR detection reagent, and the commercial PCR detection reagent is preferably TaqMan™ 2.5X Lyo-Ready 1-Step Master Mixes With Excipient reagent produced by Thermo Fisher Scientific.

[0011] The third aspect of the present invention provides a freeze-dried kit for detecting EV71, CA16 and universal enterovirus, comprising the primer-probe combination described in the first aspect. Furthermore, the primer-probe combination is prepared into a freeze-dried reagent using the method described in the first aspect.

[0012] The fourth aspect of the present invention provides a method for detecting EV71, CA16 and universal enterovirus for non-disease diagnosis purposes using the kit described in the second aspect, comprising the following steps: S1. Extracting nucleic acid from the sample to be tested; S2, directly add the sample nucleic acid obtained in S1 to the freeze-dried reagent, vortex mix, centrifuge and place in a PCR detector for detection; S3. Analysis of test results.

[0013] In some embodiments of the present invention, the reaction procedure of the PCR detector is: 45-50℃ 4-5min one cycle, 90-95℃ 1-2min one cycle, 95-98℃ 2-3S 60-62℃4-5S five cycles, 95-98℃ 2-3S 58-60℃ 4-5S for forty cycles.

[0014] In some preferred embodiments of the present invention, the reaction program of the PCR detector is: 50°C 5min for one cycle, 95°C 2min for one cycle, 98°C 3S 62°C 5S for five cycles, and 98°C 3S 60°C 5S for forty cycles.

[0015] In some embodiments of the present invention, the detection result analysis method is as follows: (1) Confirm that the Ct value of the fluorescence channel of the human internal standard is ≤31, otherwise the PCR reaction is considered invalid and needs to be retested; (2) If the Ct value of the fluorescence channel of the human internal standard is ≤31, then: A. In the sample to be tested, if the fluorescence Ct value of all probes of EV71, CA16 and universal enterovirus is greater than 36 or there is no typical S-shaped amplification curve, it is judged as a negative sample; B. If the fluorescence Ct value of the probe for universal enterovirus of the sample to be tested is ≤36, and the fluorescence Ct value of the probe for EV71 and CA16 is >36, it is judged as a positive sample for enterovirus other than CA16 and EV71; C. If the fluorescence Ct value of the probe for universal enterovirus of the sample to be tested is ≤36, and the fluorescence Ct value of the probe for EV71 is ≤36, and the fluorescence Ct value of the probe for CA16 is >36, the sample is judged as EV71 positive; D. If the fluorescence Ct value of the universal enterovirus probe of the sample to be tested is ≤36, the fluorescence Ct value of the CA16 probe is ≤36, and the fluorescence Ct value of the EV71 probe is >36, the sample is judged to be CA16 positive.

[0016] In some embodiments of the present invention, the samples to be tested are stool samples and throat swab samples.

[0017] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: The present invention is based on real-time fluorescence quantitative RT-PCR technology and is used for in vitro detection of EV71, CA16 and universal enterovirus in human fecal samples and throat swab samples, wherein the universal enterovirus includes four genotypes A, B, C, and D; and the reagent is in a freeze-dried form, and when used, there is no need to prepare PCR reaction solution, and nucleic acid can be directly added, which is easy to operate and can be stored and transported at room temperature, and the detection time is controlled to about 40 minutes, thereby improving the convenience and efficiency of the detection. At the same time, it has good specificity and sensitivity, which helps to provide intermediate reference data for clinical implementation of early diagnosis and timely treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows the screening amplification curves of three sets of primer-probe combinations designed for each detection target in the present invention; wherein the red one is primer-probe combination 1, the yellow one is primer-probe combination 2, and the blue one is primer-probe combination 3; Figure 2 A comparison chart of the amplification efficiency test results of two groups of different PCR detection reagents of the present invention is shown; wherein, blue: Lyo-Ready qPCR Mix reagent produced by Meridian, red: TaqMan™ Lyo-readyqPCR Master Mix reagent produced by Thermo Fisher Scientific; Figure 3 A comparison diagram of the appearance of freeze-dried reagents prepared by two groups of different freeze-drying procedures of the present invention is shown; Figure 4A comparison chart showing the PCR performance test of freeze-dried reagents prepared by two different freeze-drying procedures of the present invention; wherein red is procedure 1 and blue is procedure 2; Figure 5 The sensitivity test results at different copy numbers in Example 6 of the present invention are shown; Figure 6 The stability test result diagram in Example 8 of the present invention is shown; wherein, blue is channel 1, channel 1 is FAM fluorescence (universal enterovirus); green is channel 2, channel 2 is HEX fluorescence (EV71); orange is channel 3, channel 3 is ROX fluorescence (CA16); red is channel 4, channel 4 is CY5 fluorescence (human internal standard); Figure 7 The test results of 6 clinical samples in Example 9 of the present invention are shown, wherein blue is channel 1, channel 1 is FAM fluorescence (universal enterovirus); green is channel 2, channel 2 is HEX fluorescence (EV71); orange is channel 3, channel 3 is ROX fluorescence (CA16); red is channel 4, channel 4 is CY5 fluorescence (human internal standard). DETAILED DESCRIPTION

[0019] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0021] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0022] Example 1 Primer probe design and screening The enterovirus genome is a single-stranded positive-strand RNA with a total length of about 7.2 to 8.4 kb, including 5'UTR, 3'UTR and polyadenosine tail (polyA) at both ends and P1, P2 and P3 coding regions in the middle; among them, 5'UTR has a very high homology in enterovirus and is often used as a target gene for universal enterovirus detection; and the VP1 gene in the middle P1 coding region has good specificity and is often used for enterovirus genotyping; therefore, the present invention designs primer probes for 5'UTR for universal enterovirus detection, which includes four genotypes A, B, C and D, and designs primer probes for VP1 gene for EV71 and CA16 detection. Among them, universal enterovirus is labeled with FAM fluorescence, EV71 is labeled with HEX fluorescence, and CA16 is labeled with ROX fluorescence; at the same time, primer probes are designed for human β-actin gene, and CY5 fluorescence is used as the internal standard of the kit to monitor the sampling and extraction process.

[0023] According to the above scheme, three sets of primer probes were designed for universal enterovirus, CA16, EV71 and internal standard, respectively. The three sets of primer probes are shown in Table 1.

[0024] Table 1 Information of three sets of primers and probes

[0025] Note: Among the above primer probes, the 5' end of the universal enterovirus probe is modified with a FAM fluorescent group, and the 3' end is modified with a BHQ1 quencher group; the 5' end of the EV71 probe is modified with a HEX fluorescent group, and the 3' end is modified with a BHQ1 quencher group; the 5' end of the CA16 probe is modified with a ROX fluorescent group, and the 3' end is modified with a BHQ2 quencher group; the 5' end of the human internal standard probe is modified with a CY5 fluorescent group, and the 3' end is modified with a BHQ2 quencher group.

[0026] The above three sets of primer-probe combinations were used to compare the amplification efficiency. The amplification curves are shown in Figure 1 shown.

[0027] The results showed that the detection Ct value of combination 1 was relatively high among the three combinations, indicating that combination 1 had the best amplification effect. Therefore, combination 1 was selected as the final primer probe sequence.

[0028] The specific information of the primer and probe sequences of the screened universal enterovirus, CA16, EV71 and human internal standard is shown in Table 2 below.

[0029] Table 2 Primer probe sequence information

[0030] Example 2 Optimization of PCR detection reagents The Lyo-Ready 1-step qPCR Mix produced by Meridian was selected for comparison with the TaqMan™ 2.5X Lyo-Ready 1-Step Master Mixes With Excipient produced by Thermo Fisher Scientific. The detection indicators were amplification efficiency and detection limit. The results are as follows: Figure 2 shown.

[0031] The results showed that the amplification efficiency and detection limit of the three fluorescence channels of FAM, HEX and ROX were slightly better than those of the Meridian reagent. Therefore, the Thermo Fisher reagent was selected as the PCR detection reagent of the present invention.

[0032] Example 3 Optimization of freeze-drying process In the freeze-drying process, the temperature and time of the sublimation stage have an important influence on the morphology and performance of the freeze-dried reagents. Therefore, this experiment optimizes the temperature and time of the freeze-drying sublimation stage. The freeze-drying sublimation temperature and time are negatively correlated. This experiment set two freeze-drying programs for the sublimation stage as shown in Table 3. By comparing the morphology and PCR performance of the freeze-dried reagents under different freeze-drying programs, the optimal freeze-drying program was selected.

[0033] Table 3 Freeze-drying procedure

[0034] The freeze-dried reagents obtained by the two freeze-drying procedures are as follows Figure 3 shown.

[0035] The results showed that there was no significant difference in the morphology of the freeze-dried reagents obtained by the two freeze-drying procedures. Both were complete and full, without shrinkage, collapse, or remelting, and met the requirements.

[0036] The freeze-dried reagents obtained from the two freeze-drying procedures were subjected to comparative tests on PCR performance, and the test indicators included amplification efficiency and detection limit. The results are as follows: Figure 4 shown.

[0037] The results showed that the amplification efficiency and detection limit of the three fluorescence channels of FAM, HEX and ROX were better than those of freeze-drying procedure 2, so freeze-drying procedure 1 was selected as the freeze-drying procedure of the present invention.

[0038] Example 4 Preparation of lyophilized reagent 1. Dilution of primers and probes Primer probe powder is diluted to 20 μM with 1×TE Buffer. The calculation method is as follows: Dilute according to the number of nmoles: 1×TE Buffer added to each tube = number of nmoles × 50 (μL); after adding 1×TE Buffer, vortex for about 30 seconds to evenly mix, and briefly centrifuge at low speed.

[0039] 2. Preparation of primer and probe working solution After the primer and probe are diluted, prepare the primer and probe working solution according to Table 4.

[0040] Table 4 Composition of primer and probe working solution

[0041] 3. Preparation of PCR premix The lyophilizable PCR detection reagent used in the present invention is TaqMan™ 2.5X Lyo-Ready 1-Step Master Mixes With Excipient reagent produced by Thermo Fisher Scientific (the reagent is a commercially available product, and the main components of the mixed solution include detection buffer, reverse transcriptase, DNA polymerase, lyophilization additives, etc.), and the preparation of the PCR premix is ​​shown in Table 5.

[0042] Table 5 Preparation of PCR premix

[0043] 4. Freeze-drying The PCR premix prepared as described above was dispensed into the bottom of the PCR reaction tube (96-well plate or eight-well tube) at 15 μL / well. After the dispensing was completed, it was placed in a freeze dryer and freeze-dried according to freeze-drying program 1 in Table 3; thus, the freeze-dried reagent for detecting EV71, CA16 and universal enterovirus was obtained.

[0044] The freeze-dried reagent kit for detecting EV71, CA16 and universal enterovirus contains the above freeze-dried reagent.

[0045] Example 5 Method for detecting EV71, CA16 and universal enterovirus using a kit (1) Extract the sample to be tested and prepare nucleic acid According to the instructions of the nucleic acid extraction or purification reagent produced by Jiangsu Merle Biotechnology Co., Ltd. (Registration Number: Sutai Xiebei 20180121), extract the sample to be tested and prepare nucleic acid; applicable sample types include human fecal samples and throat swab samples.

[0046] (2) PCR testing The extracted nucleic acid was added to the freeze-dried reagent in Example 4 at 25 μL / well, vortexed and briefly centrifuged to ensure that the freeze-dried reagent was completely dissolved and did not contain obvious bubbles; after the addition was completed, the sample was transferred to the detection area for on-machine detection.

[0047] Place the PCR reaction tube after sample addition into the fluorescent PCR detector, set the PCR reaction program for detection as shown in Table 6. The collection of fluorescent signals is set to FAM, HEX, ROX and CY5, the data collection is set at 60°C, and the reaction volume of each well is set to 25 μL.

[0048] Table 6 PCR reaction program

[0049] (3) Results analysis After the reaction is completed, the instrument automatically saves the results. After analyzing the image, adjust the Start value, End value and Threshold value of the Baseline (you can adjust it yourself, the Start value can be between 3-15, and the End value can be between 5-20). The Ct value of the CY5 fluorescence channel (human internal standard) should be ≤31, otherwise the PCR reaction is considered invalid and needs to be retested. If the above conditions are met, analyze according to the following method: (1) When the FAM, HEX, or ROX fluorescence Ct value of the sample to be tested is greater than 36 or there is no typical S-shaped amplification curve, it is judged as a negative sample; (2) If the FAM fluorescence Ct value of the sample to be tested is ≤36, and the HEX and ROX fluorescence Ct values ​​are >36, it is judged to be a positive sample for other enteroviruses other than CA16 and EV71; (3) If the FAM fluorescence Ct value of the sample to be tested is ≤36, the HEX fluorescence Ct value is ≤36, and the ROX fluorescence Ct value is >36, it is judged as an EV71 positive sample; (4) If the FAM fluorescence Ct value of the sample to be tested is ≤36, the ROX fluorescence Ct value is ≤36, and the HEX fluorescence Ct value is >36, it is judged as a CA16 positive sample.

[0050] Example 6 Sensitivity Test In vitro transcribed RNA of universal enterovirus (CA6), CA16 and EV71 were selected as simulated samples for the detection limit test. The specific scheme is as follows: in vitro transcribed RNA with known copy number was diluted to 400 copies / mL, 200 copies / mL and 100 copies / mL respectively with enterovirus negative samples. Each in vitro transcribed RNA was extracted and tested 10 times repeatedly. The lowest concentration with a positive detection rate of 95% was taken as the minimum detection limit. The test results are shown in Table 7. The amplification curves of the minimum detection limits of universal enterovirus, CA16 and EV71 are shown in Table 7. Figure 5 shown.

[0051] Table 7 Detection limit detection Ct value results

[0052] The results showed that the minimum detection limits of universal enterovirus, CA16 and EV71 could reach 200 copies / mL.

[0053] Example 7 Specificity Test Cultures of other pathogens with the same infection site as the enterovirus or similar infection symptoms were selected for detection using the reagent of the present invention, and each pathogen culture was sequenced and confirmed. The results are shown in Table 8.

[0054] Table 8 Specificity detection Ct value results

[0055] The results showed that none of the 12 other pathogen cultures were amplified, indicating that the present invention had good specificity.

[0056] Example 8 Stability Test The freeze-dried reagents were stored at room temperature (20℃~25℃) and taken out at the 6th and 12th months of storage. CA16 samples with four different concentrations of 200 copies / mL, 2000 copies / mL, 20000 copies / mL, and 200000 copies / mL and EV71 samples with four different concentrations of 400 copies / mL, 4000 copies / mL, 40000 copies / mL, and 400000 copies / mL were used for detection. The detection indicators included amplification efficiency and detection limit. Among them, the detection limit detection was carried out in 8 groups of parallel tests using 200 copies / mL of CA16 samples and 400 copies / mL of EV71 samples. The test results were compared with the results of freeze-dried samples out of the box (month 0). The comparison results are shown in Tables 9-10. The amplification curves are shown in Tables 9-10. Figure 6 shown.

[0057] Table 9 Stability--Ct value results of amplification efficiency detection

[0058] Table 10 Stability--Ct value results of detection limit detection

[0059] The results showed that there was no significant difference between the Ct values ​​of the samples stored at room temperature for 6 months and 12 months and the Ct values ​​of the samples tested out of the box. This shows that the freeze-dried reagent of the present invention can be stably stored at room temperature for 12 months.

[0060] Example 9 Clinical Sample Detection Six clinical samples were selected: sample 1, sample 2, sample 3, sample 4, sample 5, and sample 6, and the detection method of the present invention was used. The detection Ct values ​​are shown in Table 11, and the amplification curves are shown in Figure 7 shown.

[0061] Table 11 Ct value results of clinical sample detection

[0062] The results show that according to the Ct value results detected by different fluorescence channels, the sample detection results can be successfully determined according to the result analysis method in Example 5.

[0063] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A primer-probe combination for detecting EV71, CA16 and universal enterovirus, characterized in that: The primer-probe combination comprises: (1) The primer pair for targeted amplification of universal enterovirus is shown in SEQ ID NO.1-SEQ ID NO.2; the probe for detecting universal enterovirus is shown in SEQ ID NO.3; the universal enterovirus includes four genotypes: A, B, C, and D; (2) The primer pairs for targeted amplification of EV71 are shown in SEQ ID NO.4-SEQ ID NO.5, SEQ ID NO.7-SEQ ID NO.8; the probes for detecting EV71 are shown in SEQ ID NO.6 and SEQ ID NO.9; (3) The primer pairs for targeted amplification of CA16 are shown as SEQ ID NO.10-SEQ ID NO.11 and SEQ ID NO.12-SEQ ID NO.13; the probe for detecting CA16 is shown as SEQ ID NO.

14.

2. The primer-probe combination for detecting EV71, CA16 and universal enterovirus according to claim 1, characterized in that: Also includes: The primer pair for targeted amplification of the human internal standard is shown in SEQ ID NO.15-SEQ ID NO.16; The probe for detecting the human internal standard is shown in SEQ ID NO.

17.

3. The primer-probe combination for detecting EV71, CA16 and universal enterovirus according to claim 2, characterized in that: In the primer-probe combination, the 5' end of the probe is provided with a fluorescent group, and the 3' end is provided with a quenching group. The fluorescent groups on the probes for detecting the same target are the same, and the fluorescent groups on the probes for different targets are different.

4. The primer-probe combination for detecting EV71, CA16 and universal enterovirus according to claim 3, characterized in that: The fluorescent group is one of FAM, HEX, VIC, ROX and CY5, and the quenching group is one of BHQ1, MGB and BHQ2.

5. A method for preparing a freeze-dried reagent for detecting EV71, CA16 and universal enterovirus, characterized in that: The steps include: (1) Preparation of primer-probe working solution: The primer-probe dry powder in the primer-probe combination of any one of claims 1 to 4 is first diluted with TE Buffer respectively, and then the primer-probe working solution is prepared: the final concentration of the primer pair for amplifying universal enterovirus is 1 μM, and the final concentration of the probe is 0.4 μM; the final concentration of the primer pair for amplifying EV71 is 0.8 μM, and the final concentration of the probe is 0.4 μM; the final concentration of the primer pair for amplifying CA16 is 0.4 μM, and the final concentration of the probe is 0.2 μM; the final concentration of the primer pair for amplifying human internal standard is 0.4 μM, and the final concentration of the probe is 0.3 μM; (2) Prepare PCR premix: Mix the primer probe working solution and PCR detection reagent in a ratio of 1:2 to obtain a PCR premix; (3) Freeze-drying procedure: The PCR premix is ​​divided into aliquots and freeze-dried using the following freeze-drying procedure to obtain the freeze-dried reagent: A. Pre-freezing: plate temperature -45°C, cooling time 60min, holding time 120min; B. Sublimation drying: board temperature -25℃, heating time 60min, hold time 600min, pressure 10Pa; C. Analytical drying: plate temperature 30°C, heating time 120min, hold time 120min, pressure 10Pa.

6. A freeze-dried kit for detecting EV71, CA16 and universal enterovirus, characterized in that: The invention comprises the primer-probe combination according to any one of claims 1 to 4.

7. A freeze-dried kit for detecting EV71, CA16 and universal enterovirus, characterized in that: A freeze-dried reagent prepared by the method according to claim 5.

8. A method for detecting EV71, CA16 and universal enterovirus for non-disease diagnosis purposes using the kit according to claim 6, characterized in that: The steps include: S1. Extracting nucleic acid from the sample to be tested; S2, directly add the sample nucleic acid obtained in S1 to the freeze-dried reagent, vortex mix, centrifuge and place in a PCR detector for detection; S3, analysis of test results; The reaction procedure of the PCR detector is: 45-50℃ 4-5min one cycle, 90-95℃ 1-2min one cycle, 95-98℃ 2-3S 60-62℃ 4-5S five cycles, 95-98℃ 2-3S 58-60℃ 4-5S for forty cycles.

9. The method according to claim 8, characterized in that: The test result analysis method is as follows: (1) Confirm that the Ct value of the fluorescence channel of the human internal standard is ≤31, otherwise the PCR reaction is considered invalid and needs to be retested; (2) If the Ct value of the fluorescence channel of the human internal standard is ≤31, then: A. In the sample to be tested, if the fluorescence Ct value of all probes of EV71, CA16 and universal enterovirus is greater than 36 or there is no typical S-shaped amplification curve, it is judged as a negative sample; B. If the fluorescence Ct value of the probe for universal enterovirus of the sample to be tested is ≤36, and the fluorescence Ct value of the probe for EV71 and CA16 is >36, it is judged as a positive sample for enterovirus other than CA16 and EV71; C. If the fluorescence Ct value of the probe for universal enterovirus of the sample to be tested is ≤36, and the fluorescence Ct value of the probe for EV71 is ≤36, and the fluorescence Ct value of the probe for CA16 is >36, the sample is judged as EV71 positive; D. If the fluorescence Ct value of the universal enterovirus probe of the sample to be tested is ≤36, the fluorescence Ct value of the CA16 probe is ≤36, and the fluorescence Ct value of the EV71 probe is >36, the sample is judged to be CA16 positive.

10. The method according to claim 8, characterized in that: The samples to be tested are stool samples and throat swab samples.

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