Enterovirus D68 detection method and detection kit thereof

Abstract

The invention discloses an enterovirus D68 detection method and a detection kit thereof, and belongs to the technical field of molecular biology. The method comprises the following steps: collecting a sample, and extracting nucleic acid to obtain a pretreated sample; mixing and reacting with an EV-D68 primer probe composition to obtain a nested MIRA amplification detection system, and detecting; the EV-D68 primer probe composition comprises the following components: a peripheral MIRA amplification primer, an internal MIRA amplification primer and a detection probe. The invention aims to provide an enterovirus D68 detection method and a detection kit thereof, based on a nested MIRA detection system, an EV-D68 rapid nucleic acid detection method is established for the first time, and the EV-D68 rapid nucleic acid detection method comprises two forms of fluorescence detection and colloidal gold test strip detection. The method has the advantages of rapidness, high sensitivity, strong specificity, low cost and the like, and is suitable for clinically and rapidly diagnosing pathogens.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and specifically relates to an enterovirus D68 detection method and a detection kit thereof. Background Art

[0002] Enterovirus (EV) is a class of single-stranded positive-sense RNA viruses belonging to the genus Enterovirus of the family Picornaviridae. The genus Enterovirus includes many types, such as human enterovirus A to D, rhinovirus A to C, and enterovirus E to L that infect non-human hosts. Enterovirus D68 (EV-D68), as a species in this genus, has caused multiple outbreaks worldwide since 2014 and has become a re-emerging type of infection, especially in children. EV-D68 infection can cause serious health problems such as asthma-like illness, severe respiratory diseases and acute asthenic myelitis.

[0003] Since the symptoms of EV-D68 are similar to those of other respiratory viral infections, accurate and rapid diagnosis is crucial for clinical treatment decisions and controlling the spread of the virus. Currently, real-time RT-PCR technology is considered the "gold standard" for diagnosing EV-D68 infection, but this method has problems such as insufficient sensitivity, complex operation and long time consumption. The positive rate is only 14%-50%, which cannot meet the clinical demand for rapid detection of EV-D68. With the increase in EV-D68 genetic diversity, the effectiveness of existing detection methods is facing challenges. Therefore, the development of more reliable nucleic acid detection technology to adapt to the genetic variation of the virus has become a research focus.

[0004] In the field of isothermal amplification technology, multi-enzyme isothermal rapid amplification (MIRA) technology has attracted attention due to its high amplification efficiency, stability and anti-interference ability. MIRA technology relies on multiple functional proteins (such as helicase, recombinase, single-strand binding protein, DNA polymerase, etc.) to achieve rapid amplification of nucleic acids at room temperature, and is characterized by being fast, simple and accurate. Compared with other isothermal amplification technologies such as RPA and LAMP, MIRA technology exhibits higher amplification efficiency and stability. Although MIRA technology has been used to detect a variety of pathogens, there have been no reports on the use of MIRA for enterovirus D68 detection. Summary of the invention

[0005] The present invention aims to provide an enterovirus D68 detection method and a detection kit thereof. Based on the Nest MIRA detection system, a rapid nucleic acid detection method for EV-D68 is established for the first time, including two forms of fluorescence detection and colloidal gold test strip detection. This method has the advantages of rapidity, high sensitivity, strong specificity, and low cost, and is suitable for rapid clinical diagnosis of pathogens.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for detecting enterovirus D68 for non-disease diagnosis and / or treatment purposes, the detection method comprising the following steps: S1. Sample collection: Use a clean throat swab, gently insert it into the mouth, wipe the posterior pharyngeal wall and tonsil area, and rotate it to fully collect secretions; S2, nucleic acid extraction: adding the secretion obtained in step S1 into an RNA releasing preservative to obtain a pretreated sample; S3, nested MIRA amplification: the pre-treated sample obtained in step S2 is mixed with the EV-D68 primer-probe combination to obtain the nested MIRA amplification detection system for detection; The EV-D68 primer-probe composition comprises the following components: a peripheral MIRA amplification primer pair, an internal MIRA amplification primer pair and a detection probe.

[0007] Preferably, in step S3, the pretreated sample obtained in step S2 is used as a template, a peripheral MIRA amplification primer pair is added, the reaction is carried out at 39°C for 10 min, centrifuged at 1000 rpm for 10 s, mixed with the inner MIRA amplification primer pair loaded on the centrifuge tube cap, and reacted at 39°C for 20 min to obtain the nested MIRA amplification detection system.

[0008] Preferably, the peripheral MIRA amplification primer pair is one of the following: (1) a primer pair consisting of EV-D68-OF-1 as shown in SEQ ID NO: 1 and EV-D68-OR-1 as shown in SEQ ID NO: 3; (2) a primer pair consisting of EV-D68-OF-1 as shown in SEQ ID NO: 1 and EV-D68-OR-2 as shown in SEQ ID NO: 4; (3) EV-D68-OF-2 as shown in SEQ ID NO: 2 and EV-D68-OR-1 as shown in SEQ ID NO: 3; (4) EV-D68-OF-2 as shown in SEQ ID NO: 2 and EV-D68-OR-2 as shown in SEQ ID NO: 4; The inner MIRA amplification primer pair is one of the following: (1) EV-D68-IF-1 as shown in SEQ ID NO:5 and EV-D68-IR-1 as shown in SEQ ID NO:7; (2) EV-D68-IF-1 as shown in SEQ ID NO:5 and EV-D68-IR-2 as shown in SEQ ID NO:8; (3) EV-D68-IF-2 as shown in SEQ ID NO:6 and EV-D68-IR-1 as shown in SEQ ID NO:7; (4) EV-D68-IF-2 as shown in SEQ ID NO:6 and EV-D68-IR-2 as shown in SEQ ID NO:8.

[0009] Preferably, when the detection probe is a fluorescent detection probe, the detection method is fluorescent detection, wherein the downstream primer EV-D68-IR-1 of the inner MIRA amplification primer pair used is not labeled with biotin, and the sequence of the fluorescent detection probe is as shown in SEQ ID NO:9.

[0010] Preferably, the detection probe is a colloidal gold test strip detection probe, and the detection method is colloidal gold test strip detection, wherein the downstream primer EV-D68-IR-1 of the inner MIRA amplification primer pair used is labeled with biotin, and its 5' end is modified with biotin, and the colloidal gold test strip detection probe sequence is shown in SEQ ID NO:10.

[0011] The present invention also provides an EV-D68 primer-probe combination for enterovirus D68 detection, comprising a peripheral MIRA amplification primer pair, an inner MIRA amplification primer pair and a detection probe; the peripheral MIRA amplification primer pair is one of the following: (1) a primer pair consisting of EV-D68-OF-1 as shown in SEQ ID NO: 1 and EV-D68-OR-1 as shown in SEQ ID NO: 3; (2) a primer pair consisting of EV-D68-OF-1 as shown in SEQ ID NO: 1 and EV-D68-OR-2 as shown in SEQ ID NO: 4; (3) EV-D68-OF-2 as shown in SEQ ID NO: 2 and EV-D68-OR-1 as shown in SEQ ID NO: 3; (4) EV-D68-OF-2 as shown in SEQ ID NO: 2 and EV-D68-OR-2 as shown in SEQ ID NO: 4; The inner MIRA amplification primer pair is one of the following: (1) EV-D68-IF-1 as shown in SEQ ID NO:5 and EV-D68-IR-1 as shown in SEQ ID NO:7; (2) EV-D68-IF-1 as shown in SEQ ID NO:5 and EV-D68-IR-2 as shown in SEQ ID NO:8; (3) EV-D68-IF-2 as shown in SEQ ID NO:6 and EV-D68-IR-1 as shown in SEQ ID NO:7; (4) EV-D68-IF-2 as shown in SEQ ID NO:6 and EV-D68-IR-2 as shown in SEQ ID NO:8; The detection probe is a fluorescent detection probe or a colloidal gold test strip detection probe; The fluorescent detection probe sequence is shown in SEQ ID NO:9, and the colloidal gold test strip detection probe sequence is shown in SEQ ID NO:10.

[0012] The present invention also provides the use of the EV-D68 primer-probe combination in the preparation of a product for detecting enterovirus D68.

[0013] The present invention also provides the use of the EV-D68 primer-probe combination in preparing a product for identifying or assisting in identifying enterovirus D68.

[0014] The present invention also provides an enterovirus D68 detection kit, which comprises an RNA constant temperature rapid amplification reagent and the EV-D68 primer probe composition.

[0015] Preferably, the RNA isothermal rapid amplification reagent is a fluorescent type or a colloidal gold type.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: (1) Based on the Nest MIRA detection system, the present invention established for the first time a rapid nucleic acid detection method for EV-D68 (including fluorescence detection and colloidal gold test strip detection). This method has the advantages of rapidity, high sensitivity, strong specificity, and low cost, and is suitable for rapid clinical diagnosis of pathogens.

[0017] (2) Compared with traditional methods for clinical diagnosis of EV-D68 such as virus isolation and culture, RT-PCR / qRT PCR nucleic acid detection and serological detection, the present invention has more advantages and meets the requirements of POCT (Point-of Care Testing, i.e., rapid, sensitive, specific, low equipment requirements and economical). In addition, EV-D68 detection can be completed within 35-50 minutes, and the detection limit is 0.5 copy / μL.

[0018] (3) Compared with traditional viral nucleic acid detection technology, this method does not require expensive detection equipment. Only a metal bath set at 39°C is needed to complete nucleic acid amplification; the detection process only requires the use of a portable fluorescence reader, and even when using colloidal gold test strips for detection, no additional equipment is required. This greatly reduces the requirements for equipment and personnel operating skills.

[0019] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the EV-D68 detection method of the nested MIIRA detection system of the present invention, wherein Figure 1 A in the figure is the EV-D68 detection process. Figure 1 B in the figure is a schematic diagram of nested MIRA amplification and detection; Figure 2 The inner primer optimization result provided in Example 1; Figure 3 The peripheral primer optimization results provided in Example 1; Figure 4 The reaction temperature optimization result of Example 1; Figure 5 The sensitivity fluorescence detection test results of Nest's MIRA in Example 1; Figure 6 The sensitivity colloidal gold test strip test results of Nest's MIRA in Example 1; Figure 7 These are the test results for consistency evaluation of the nested MIRA and RT-PCR detection methods in Example 2 and Comparative Example 1. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0022] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0023] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0024] Example 1 This example provides an EV-D68 primer-probe combination for enterovirus D68 detection, as follows: Design of primers and probes for multi-enzyme isothermal amplification 1. Download 100 EV-D68 full-length gene reference sequences from the NCBI website, including EV-D68 (A1, A2, B1, B2, B3, C subtypes), and integrate them into a TXT document.

[0025] 2. Use Mega 7 software to perform multiple sequence alignment of nucleotide sequences, and select the conserved region of VP1 gene in the EV-D68 typing region for primer and probe design. Use Primer Select software to assist in the design, avoid the hairpin structure and dimer between primers, so as to optimize the detection performance of primers and probes, and obtain the EV-D68 primer-probe combination.

[0026] The sequences of the primer-probe combinations for nested MIRA amplification of EV-D68 are shown in Table 1 .

[0027] Table 1 EV-D68 primer-probe combination sequence ;

[0028] Note: / i6FAMdT / means "T base is modified by 5'6-FAM fluorescence"; / idSp / means "tetrahydrofuran modification site"; / iBHQ1dT / means "T base is modified by BHQ1 quencher group"; 3'C3 Spacer means "blocking group".

[0029] The EV-D68 primer-probe combination comprises a peripheral MIRA amplification primer pair, an inner MIRA amplification primer pair and a detection probe; the peripheral MIRA amplification primer pair is one of the following: (1) a primer pair consisting of EV-D68-OF-1 as shown in SEQ ID NO: 1 and EV-D68-OR-1 as shown in SEQ ID NO: 3; (2) a primer pair consisting of EV-D68-OF-1 as shown in SEQ ID NO: 1 and EV-D68-OR-2 as shown in SEQ ID NO: 4; (3) EV-D68-OF-2 as shown in SEQ ID NO: 2 and EV-D68-OR-1 as shown in SEQ ID NO: 3; (4) EV-D68-OF-2 as shown in SEQ ID NO: 2 and EV-D68-OR-2 as shown in SEQ ID NO: 4; The inner MIRA amplification primer pair is one of the following: (1) EV-D68-IF-1 as shown in SEQ ID NO:5 and EV-D68-IR-1 as shown in SEQ ID NO:7; (2) EV-D68-IF-1 as shown in SEQ ID NO:5 and EV-D68-IR-2 as shown in SEQ ID NO:8; (3) EV-D68-IF-2 as shown in SEQ ID NO:6 and EV-D68-IR-1 as shown in SEQ ID NO:7; (4) EV-D68-IF-2 as shown in SEQ ID NO:6 and EV-D68-IR-2 as shown in SEQ ID NO:8; The detection probe is a fluorescent detection probe or a colloidal gold test strip detection probe; The fluorescent detection probe sequence is shown in SEQ ID NO:9, and the colloidal gold test strip detection probe sequence is shown in SEQ ID NO:10.

[0030] Sample nucleic acid extraction and preparation of viral template 1. Use the viral DNA / RNA extraction kit to extract EV-D68 viral RNA, and use specific primers for one-step RT-PCR amplification. The specific steps are as follows: (1) Extraction of EV-D68 viral RNA: Take 200 μL of inactivated EV-D68 virus storage solution and use a viral DNA / RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP315) to extract viral RNA according to the kit operating instructions to obtain a total of 50 μL of RNA template.

[0031] (2) RT-PCR amplification: Use a one-step RT-PCR kit for PCR reaction.

[0032] Prepare the following reaction system: Reaction buffer: 20 μL, RT-PCR reaction enzyme: 2 μL, upstream primer D68-F1 (5'-TRGCAGCAAARGAYGAYTT-3' (SEQ ID NO.11)) (10 μM): 2 μL, downstream primer D68-R1 (5'-CKGAYTGCCARTGGAATGARTCYTG-3' (SEQ ID NO.12)) (10 μM): 2 μL, enzyme-free water: 9 μL, RNA template: 5 μL.

[0033] Amplification procedure: (1) Reverse transcription: 50°C, 30 min; (2) Pre-denaturation: 94°C, 1 min; (3) PCR amplification: 94°C, 30 s; 56°C, 30 s; 72°C, 1 min, 40 cycles.

[0034] 2. Perform 2% agarose gel electrophoresis on the PCR product and purify the amplified product using a DNA product purification kit; 3. Subclone the VP1 gene of EV-D68 into the pUC57 vector using the TA cloning kit (TaKaRa). Use a plasmid DNA extraction kit to extract plasmid DNA, quantify its initial concentration using a UV spectrophotometer, and dilute pUC57-EV-D68-VP1 to 5×10 3 copies / μL, and the EV-D68 positive clone plasmid was obtained for later use.

[0035] The information of each bacteria / strain used in the present invention is shown in Table 2.

[0036] Table 2 Information of each bacteria / strain ;

[0037] The effects of the peripheral MIRA amplification primer pair and the inner MIRA amplification primer pair provided in Example 1 were verified by the following experiments: 1. Screening of primer pairs for in-line MIRA amplification: The optimal in-line MIRA amplification primer pairs were screened: (1) EV-D68-IF-1 and EV-D68-IR-1; (2) EV-D68-IF-1 and EV-D68-IR-2; (3) EV-D68-IF-2 and EV-D68-IR-1 (4) EV-D68-IF-2 and EV-D68-IR-2. According to the instructions of the RNA Constant Temperature Rapid Amplification Kit (Fluorescence Type) (Anpu Future Biotechnology Co., Ltd.), the reaction system configuration is shown in Table 3: Table 3 Reaction system configuration ;

[0038] With the provided 5×10 3 The EV-D68 positive clone plasmid with 100 copies / μL was used as a template and reacted at 40°C for 20 minutes. During this period, a fluorescence quantitative PCR instrument or a convenient fluorescence reader was used to read the fluorescence every 30 seconds. Figure 2 .

[0039] Depend on Figure 2 It can be seen that the fluorescence value of the amplification curve of the inner MIRA amplification primer pair (1) EV-D68-IF-1 and EV-D68-IR-1 is higher than that of the other three combinations when the reaction cycle number is 40, that is, this primer combination has a higher amplification efficiency. Therefore, the optimal inner MIRA amplification primer pair is selected as: EV-D68-IF-1 and EV-D68-IR-1.

[0040] 2. Screening of peripheral MIRA amplification primer pairs: The optimal peripheral MIRA amplification primer pairs were screened: (1) EV-D68-OF-1 and EV-D68-OR-1; (2) EV-D68-OF-1 and EV-D68-OR-2; (3) EV-D68-OF-2 and EV-D68-OR-1; (4) EV-D68-OF-2 and EV-D68-OR-2. The four peripheral MIRA amplification primer pairs were used together with the optimal internal amplification primer pair EV-D68-IF-1 and EV-D68-IR-1 for nested MIRA detection.

[0041] Referring to the instructions of RNA Constant Temperature Rapid Amplification Kit (Fluorescence Type) (Anpu Future Biotechnology Co., Ltd.), the Nested MIRA reaction system was prepared as shown in Tables 4 and 5: Table 4 Peripheral MIRA reaction system: ;

[0042] Table 5 Inner MIRA reaction system: ;

[0043] The Nested MIRA reaction is carried out in a reaction tube. The peripheral MIRA amplification system is loaded at the bottom of the reaction tube after configuration, and the inner MIRA reaction system is loaded in the lid of the reaction tube after configuration. The reaction process adopts the 40°C recommended in the instructions of the RNA Constant Temperature Rapid Amplification Kit (Fluorescence Type). Specifically: the peripheral MIRA reacts at 40°C for 10 minutes, and the inner MIRA reaction system in the lid is mixed with the peripheral MIRA reaction system by centrifugation or flipping the reaction tube, and the reaction continues at 40°C for 20 minutes. During this period, a fluorescent quantitative PCR instrument or a convenient fluorescence reader is used to read the fluorescence every 30 seconds. The results are as follows: Figure 3 .

[0044] Depend on Figure 3 It can be seen that the fluorescence value of the amplification curve of the peripheral MIRA amplification primer pair (4) EV-D68-OF-2 and EV-D68-OR-2 is higher than that of the other three combinations when the reaction cycle number is 40, that is, this primer combination has a higher amplification efficiency. Therefore, the combination of EV-D68-OF-2 and EV-D68-OR-2 is selected as the best peripheral MIRA amplification primer.

[0045] 3. Optimization of the optimal reaction temperature The reaction temperature affects the sensitivity of the Nest MIRA detection system. Previously, the RNA Constant Temperature Rapid Amplification Kit (Fluorescence Type) was used for reaction at 40°C as recommended in the instructions, and good amplification results were achieved. However, for different pathogens, due to different detection targets and primer sequences, the optimal primer amplification reaction temperature may be different.

[0046] In the present invention, five reaction temperatures of 38°C, 39°C, 40°C, 41°C and 42°C were set to optimize the optimal reaction temperature. The reaction system was the best inner MIRA amplification primer pair screened above: EV-D68-IF-1 and EV-D68-IR-1; the outer MIRA amplification primer pair was: EV-D68-OF-2 and EV-D68-OR-2. The results are as follows Figure 4 .

[0047] Depend on Figure 4 It can be seen that the fluorescence values ​​detected when the reaction temperature is too high (41℃, 42℃) and too low (38℃) are both low; there is no significant difference in fluorescence values ​​between 39℃ and 40℃, and 39℃ is slightly higher (at 40 cycles). Therefore, 39℃ is selected as the optimal reaction temperature for Nested MIRA.

[0048] 4. Specificity and sensitivity evaluation: Specificity evaluation: In order to evaluate the specificity of the EV-D68 detection method based on the Nest MIRA detection system, 12 other genotypes of enterovirus and 8 common respiratory infection pathogens were selected in the present invention. As shown in Table 2, the cross-reaction of both the fluorescence detection and the colloidal gold test strip detection methods were evaluated.

[0049] The reaction system is the best inner MIRA amplification primer pair screened above: EV-D68-IF-1 and EV-D68-IR-1; the outer MIRA amplification primer pair: EV-D68-OF-2 and EV-D68-OR-2, and the reaction temperature is 39°C. Specifically: the outer MIRA amplification primer pair reacts at 39°C for 10 minutes, and the inner MIRA reaction system in the lid is mixed with the outer MIRA reaction system by centrifugation or flipping the reaction tube, and the reaction is continued at 39°C for 20 minutes to obtain the sample to be tested. EV-D68 is detected using fluorescence detection or colloidal gold test strips, specifically: The reagents used for fluorescence detection of EV-D68 are: RNA constant temperature rapid amplification reagent (fluorescence type); peripheral MIRA amplification primers: EV-D68-OF-2, EV-D68-OR-2; internal MIRA amplification primers: EV-D68-IF-1, EV-D68-IR-1; fluorescence detection probe; When using colloidal gold test strips to detect RV-D68, the detection reagent used is: RNA constant temperature rapid amplification reagent (colloidal gold type), the downstream primer EV-D68-IR-1 of the inner MIRA amplification primer pair used is biotin-labeled, and its 5' end is biotin-modified, colloidal gold test strip detection probe, and the rest is the same as the fluorescence detection method.

[0050] The specific detection effect of pathogens is shown in Table 6. The detection results of other common pathogens were all negative, and only the detection result of EV-D68 was positive, indicating that the present invention has good specificity and has no cross reaction with other common enterovirus and respiratory tract infection pathogens.

[0051] Table 6 Specific detection effect of pathogens ;

[0052] Sensitivity evaluation: The pUC57-EV-D68-VP1 plasmid DNA was diluted 10-fold to prepare a concentration of 5×10 5 , 5×10 4 , 5×10 3 , 5×10 2 , 5×10 1 , 5×10 0 , 0.5copy / μL positive samples. The detection method was the same as the above specificity evaluation to determine the detection limit of the EV-D68 detection method based on the Nest MIRA detection system. The results are as follows Figure 5-Figure 6 .

[0053] Depend on Figure 5 and Figure 6 It can be seen that the detection limit of the EV-D68 detection method based on the Nest MIRA detection system established by the present invention is 0.5 copy / μL (i.e. 500 copies / mL). Whether it is fluorescence detection or colloidal gold test strip detection, the same detection limit can be achieved.

[0054] Example 2 This example provides a method for detecting enterovirus D68 for non-disease diagnosis and / or treatment purposes, the detection method comprising the following steps: S1. Sample collection: 20 EV-D68 infected patients and 10 healthy people were collected. A clean throat swab was gently inserted into the oral cavity to wipe the posterior pharyngeal wall and tonsil area, and rotated to fully collect secretions. S2, nucleic acid extraction: adding the secretion obtained in step S1 into an RNA releasing preservative to obtain a pretreated sample; S3. Nested MIRA amplification: The pretreated sample obtained in step S2 was used as a template, and the target fragment was amplified using the reverse transcription multi-enzyme constant temperature amplification technology and specific peripheral MIRA primers, reacted at 39°C for 10 minutes, centrifuged at 1000 rpm for 10 seconds, mixed with the inner MIRA amplification primers loaded on the centrifuge tube cover, and reacted at 39°C for 20 minutes, wherein the outer MIRA amplification primer pair is EV-D68-OF-2 and EV-D68-OR-2, and the inner MIRA amplification primer pair is EV-D68-IF-1 and non-biotin labeled EV-D68-IR-1; the EV-D68-OF-2 sequence is shown in SEQ ID NO:2; the EV-D68-OR-2 sequence is shown in SEQ ID NO:4; the EV-D68-IF-1 sequence is shown in SEQ ID NO:5; the non-biotin labeled EV-D68-IR-1 sequence is shown in SEQ ID NO:7; the fluorescent detection probe sequence is shown in SEQ ID As shown in NO:9, the Dechao MIRA amplification detection system is used for fluorescence detection. A fluorescence quantification instrument is used to read the fluorescence signal in real time. Finally, the detection result is read according to the presence or absence of an amplification curve. The detection result is as follows Figure 7 and Table 4.

[0055] Comparative Example 1 Sample collection and nucleic acid extraction were the same as in Example 2, and nested RT-PCR detection was performed using the RT-PCR detection method.

[0056] The steps of the nested RT-PCR reaction are as follows: S1. One-step RT-PCR reaction: Preparation of reaction system: reaction buffer: 10 μL, RT-PCR reaction enzyme: 1 μL, upstream primer D68-F1 (5'-TRGCAGCAAARGAYGAYTT-3' (SEQ ID NO.11)) (10 μM): 1 μL, downstream primer D68-R1 (5'-CKGAYTGCCARTGGAATGARTCYTG-3' (SEQ ID NO.12)) (10 μM): 1 μL, enzyme-free water: 2 μL, RNA template: 5 μL.

[0057] Amplification procedure: (1) Reverse transcription: 50°C, 30 min; (2) Pre-denaturation: 94°C, 1 min; (3) PCR amplification: 94°C, 30 s; 56°C, 30 s; 72°C, 2 min, 40 cycles.

[0058] S2. Prepare two-step PCR amplification reaction: Preparation of reaction system: PCR reaction premix: 10 μL, upstream primer D68-F2 (5'-AATGCAGTTGAAACAGGTGCAAG-3' (SEQ ID NO.13)) (10 μM): 1 μL, downstream primer D68-R2 (5'-GCACCAGTRGGTACAAACATTGC-3' (SEQ ID NO.14)) (10 μM): 1 μL, enzyme-free water: 3 μL, DNA template: 5 μL.

[0059] Amplification procedure: (1) Pre-denaturation: 94°C, 1 min; (2) PCR amplification: 94°C, 30 s; 56°C, 30 s; 72°C, 1 min, 40 cycles.

[0060] S3. Gel electrophoresis detection: Perform 2% agarose gel electrophoresis on the PCR product and observe the gel image to see whether the target band with a band size of 500 bp is obtained. If so, it is judged as positive, otherwise it is negative.

[0061] S4. Positive samples were sent to Qingke Biotechnology Co., Ltd. for sequencing, and the sequencing results were subjected to Blast analysis to verify whether they were EV-D68.

[0062] The results are shown in Table 7.

[0063] Table 7 Consistency evaluation results between RT-PCR results and Nest's MIIRA test results ;

[0064] Depend on Figure 7 It can be seen that the 20 EV-D68 positive samples were all positive using the detection method of Example 2, with a positive rate of 100%; the 10 negative samples from healthy people were all negative, which was consistent with the clinical diagnosis results. Compared with the RT-PCR detection method, it has a better positive rate, with a positive rate of 90% (18 / 20).

[0065] As can be seen from Table 7, the EV-D68 detection method based on the nested MIRA detection system of the present invention has better clinical detection performance than the gold standard - RT-PCR.

[0066] In summary, the present invention provides a detection kit and detection method for EV-D68 based on the Nest MIRA technology.

[0067] The main technical advantages of the present invention include: (1) Compared with RT-PCR detection technology, the EV-D68 detection method developed based on the Nest MIRA detection system does not require expensive detection equipment, gets rid of dependence on equipment, and can realize bedside detection; (2) Compared with RT-PCR, the detection time is greatly shortened (RT-PCR requires 2-3 days), and the detection can be completed in only 35-50 minutes; (3) The EV-D68 detection method based on the Nest MIRA detection system has a detection sensitivity of 0.5 copy / μL, which is more sensitive than RT-PCR; (4) It has a wide range of clinical application scenarios, and can use both fluorescence detection and colloidal gold test strips for detection, with a wider range of applications.

[0068] In summary, the EV-D68 detection method developed by the present invention based on the Nest MIRA detection system has great value in clinical accuracy, rapid diagnosis, treatment and patient prognosis, greatly improves the detection efficiency of EV-D68, and is a diagnostic technology with good prospects.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for detecting enterovirus D68 for non-disease diagnosis and / or treatment purposes, characterized in that: The detection method comprises the following steps: S1. Sample collection: Use a clean throat swab, gently insert it into the mouth, wipe the posterior pharyngeal wall and tonsil area, and rotate it to fully collect secretions; S2, nucleic acid extraction: adding the secretion obtained in step S1 into an RNA releasing preservative to obtain a pretreated sample; S3, nested MIRA amplification: the pre-treated sample obtained in step S2 is mixed with the EV-D68 primer-probe combination to obtain the nested MIRA amplification detection system for detection; The EV-D68 primer-probe composition comprises the following components: a peripheral MIRA amplification primer pair, an internal MIRA amplification primer pair and a detection probe.

2. The detection method according to claim 1, characterized in that: In step S3, the pretreated sample obtained in step S2 is used as a template, and the peripheral MIRA amplification primer pair is added, reacted at 39°C for 10 minutes, centrifuged at 1000 rpm for 10 seconds, mixed with the inner MIRA amplification primer pair loaded on the centrifuge tube cap, and reacted at 39°C for 20 minutes to obtain the nested MIRA amplification detection system.

3. The detection method according to claim 1, characterized in that: The peripheral MIRA amplification primer pair is one of the following: (1) a primer pair consisting of EV-D68-OF-1 as shown in SEQ ID NO: 1 and EV-D68-OR-1 as shown in SEQ ID NO: 3; (2) a primer pair consisting of EV-D68-OF-1 as shown in SEQ ID NO: 1 and EV-D68-OR-2 as shown in SEQ ID NO: 4; (3) EV-D68-OF-2 as shown in SEQ ID NO: 2 and EV-D68-OR-1 as shown in SEQ ID NO: 3; (4) EV-D68-OF-2 as shown in SEQ ID NO: 2 and EV-D68-OR-2 as shown in SEQ ID NO: 4; The inner MIRA amplification primer pair is one of the following: (1) EV-D68-IF-1 as shown in SEQ ID NO:5 and EV-D68-IR-1 as shown in SEQ ID NO:7; (2) EV-D68-IF-1 as shown in SEQ ID NO:5 and EV-D68-IR-2 as shown in SEQ ID NO:8; (3) EV-D68-IF-2 as shown in SEQ ID NO:6 and EV-D68-IR-1 as shown in SEQ ID NO:7; (4) EV-D68-IF-2 as shown in SEQ ID NO:6 and EV-D68-IR-2 as shown in SEQ ID NO:

8.

4. The detection method according to any one of claims 1 to 3, characterized in that: When the detection probe is a fluorescent detection probe, the detection method is fluorescent detection, wherein the downstream primer EV-D68-IR-1 of the inner MIRA amplification primer pair used is not labeled with biotin, and the sequence of the fluorescent detection probe is shown in SEQ ID NO:

9.

5. The detection method according to any one of claims 1 to 3, characterized in that: The detection probe is a colloidal gold test strip detection probe, and the detection method is colloidal gold test strip detection, wherein the downstream primer EV-D68-IR-1 of the inner MIRA amplification primer pair used is biotin-labeled, and its 5' end is biotin-modified, and the colloidal gold test strip detection probe sequence is shown in SEQ ID NO:

10.

6. An EV-D68 primer-probe composition for detecting enterovirus D68, characterized in that: It includes a peripheral MIRA amplification primer pair, an inner MIRA amplification primer pair and a detection probe; the peripheral MIRA amplification primer pair is one of the following: (1) a primer pair consisting of EV-D68-OF-1 as shown in SEQ ID NO: 1 and EV-D68-OR-1 as shown in SEQ ID NO: 3; (2) a primer pair consisting of EV-D68-OF-1 as shown in SEQ ID NO: 1 and EV-D68-OR-2 as shown in SEQ ID NO: 4; (3) EV-D68-OF-2 as shown in SEQ ID NO: 2 and EV-D68-OR-1 as shown in SEQ ID NO: 3; (4) EV-D68-OF-2 as shown in SEQ ID NO: 2 and EV-D68-OR-2 as shown in SEQ ID NO: 4; The inner MIRA amplification primer pair is one of the following: (1) EV-D68-IF-1 as shown in SEQ ID NO:5 and EV-D68-IR-1 as shown in SEQ ID NO:7; (2) EV-D68-IF-1 as shown in SEQ ID NO:5 and EV-D68-IR-2 as shown in SEQ ID NO:8; (3) EV-D68-IF-2 as shown in SEQ ID NO:6 and EV-D68-IR-1 as shown in SEQ ID NO:7; (4) EV-D68-IF-2 as shown in SEQ ID NO:6 and EV-D68-IR-2 as shown in SEQ ID NO:8; The detection probe is a fluorescent detection probe or a colloidal gold test strip detection probe; The fluorescent detection probe sequence is shown in SEQ ID NO:9, and the colloidal gold test strip detection probe sequence is shown in SEQ ID NO:

10.

7. Use of the EV-D68 primer-probe combination as claimed in claim 6 in the preparation of a product for detecting enterovirus D68.

8. Use of the EV-D68 primer-probe combination as claimed in claim 6 in the preparation of a product for identifying or assisting in the identification of enterovirus D68.

9. An enterovirus D68 detection kit, characterized in that: The kit comprises an RNA isothermal rapid amplification reagent and the EV-D68 primer-probe composition according to claim 6.

10. An enterovirus D68 detection kit according to claim 1, characterized in that: The RNA constant temperature rapid amplification reagent is one of a fluorescent type or a colloidal gold type.

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