A primer, a detection kit and application for identifying human enterovirus group C serotype

Abstract

The present invention provides a primer for identifying the serotype of human enterovirus group C, a detection kit and an application, and belongs to the field of biotechnology. A primer for identifying the serotype of human enterovirus group C, comprising a forward primer EVCY17 with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer EVCZ17 with a nucleotide sequence as shown in SEQ ID NO:2. The present invention uses a pair of primers to quickly and accurately detect 23 serotypes of the EV-C group simultaneously, with high specificity and high sensitivity. It is simpler, faster and more accurate to identify the serotype of enterovirus group C, and has good detection operation convenience.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a human enterovirus C group serotype identification primer, a detection kit and an application. Background Art

[0002] Enterovirus (EV) belongs to the genus Enterovirus of the family Picornaviridae, including human enterovirus (HEV) and animal enterovirus. Based on genomic and biological characteristics, HEV is divided into four groups: enterovirus group A (EV-A), group B (EV-B), group C (EV-C), and group D (EV-D), each of which contains different HEV serotypes. The EV-C group includes 23 serotypes (i.e. Coxsackievirus A, CVA) 1, 11, 13, 17, 19-22, 24, EV-C 95, EV-C 96, EV-C 99, EV-C 102, EV-C 104, EV-C 105, EV-C 109, EV-C 113, EV-C116-118 and poliovirus (PV1-3). These serotypes of viruses can invade multiple systems of the human body and can cause clinical manifestations such as fever, rash, conjunctivitis, respiratory system infection, central nervous system infection, myocarditis and pericarditis. Therefore, it is impossible to distinguish which serotype the pathogenic enterovirus belongs to through clinical manifestations, and it is impossible to conduct genetic characteristics and molecular epidemiological studies on the virus.

[0003] In the prior art, the identification of enterovirus serotypes is mainly divided into two categories: one is the traditional serum neutralization test for serotype identification, and the other is the identification of serotypes by molecular biology methods and the analysis of genetic characteristics by sequence comparison. The traditional method is based on the antigenicity of enterovirus, that is, the preparation of possible new virus antisera, and the cross-neutralization test with known serotype standard strains and their antisera to identify new serotype viruses. However, this method is time-consuming and cannot respond to public health emergencies.

[0004] The EV genome is a single-stranded positive-strand RNA, in which the 5'-untranslated region (5'-UTR) gene sequence is highly conserved, making it suitable as a detection target for EV primary screening. Capsid proteins VP1 (viral protein 1), VP2, and VP4 determine the antigenicity of EV and are used for serotyping. Among them, VP1 typing completely corresponds to the serotype and is considered to be the most accurate typing target. However, the existing enterovirus serological identification methods on the market have problems such as multiple pairs of primers or multiple processes when applied to enterovirus group C, cumbersome operation, and low sensitivity. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a human enterovirus C group serotype identification primer, which uses a pair of primers to identify multiple enterovirus C group serotypes and has good detection specificity and sensitivity.

[0006] The present invention provides a human enterovirus group C serotype identification primer, comprising a forward primer EVCY17 having a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer EVCZ17 having a nucleotide sequence as shown in SEQ ID NO:2.

[0007] The invention provides a serotype identification kit for enterovirus group C, comprising primers for identifying serotypes of human enterovirus group C and RT-PCR detection reagents.

[0008] Preferably, the RT-PCR detection reagent comprises IIIOne-Step RT-PCR Systemwith Taq.

[0009] Preferably, a nucleic acid extraction reagent is also included.

[0010] Preferably, the nucleic acid extraction reagent includes components of an RNA extraction kit RNeasy Mini kit.

[0011] The present invention provides application of the human enterovirus C group serotype identification primer in preparing an enterovirus C group serotype identification kit.

[0012] Preferably, the serotypes of the enterovirus group C include serotypes 1, 11, 13, 17, 19-22, 24 in coxsackievirus, serotypes 95, 96, 99, 102, 104, 105, 109, 113 and 116-118 in enterovirus group C and PV1, PV2 and PV3 in poliovirus.

[0013] The present invention provides a method for identifying enterovirus group C serotypes for non-disease diagnosis purposes, comprising the following steps:

[0014] Extracting total RNA from samples to be tested;

[0015] Using the total RNA as a template, a one-step RT-PCR reaction is performed using the human enterovirus group C serotype identification primers to obtain an amplified product;

[0016] The amplified product is sequenced, and the obtained sequencing result is compared with the VP1 gene of each serotype of enterovirus group C. If the comparison is consistent, it means that the sample to be tested contains the corresponding serotype of enterovirus group C, otherwise it means that the sample to be tested does not contain the corresponding serotype of enterovirus group C.

[0017] Preferably, the total volume of the amplification reaction system of the one-step RT-PCR reaction is 25 μL, including 12.5 μL of 2×ReactionMix buffer, 5.5 μL of RNA free H2O, 0.5 μL of EVCY17, 0.5 μL of EVCZ17, 1.0 μL of RT / Platinum TaqMix enzyme, and 5.0 μL of template RNA;

[0018] The amplification reaction program of the one-step RT-PCR reaction is preferably 50°C for 30 min; 94°C for 2 min; 94°C for 30 s, 52°C for 40 s, 72°C for 1 min 20 s, for a total of 40 cycles; and extension at 72°C for 7 min.

[0019] Preferably, the nucleotide sequence of the VP1 gene of serotypes 1, 11, 13, 17, 19 to 22, and 24 of Coxsackievirus is shown as SEQ ID NO: 5 to SEQ ID NO: 13;

[0020] The nucleotide sequences of the VP1 genes of serotypes 95, 96, 99, 102, 104, 105, 109, 113 and 116 to 118 in the enterovirus C group are shown in SEQ ID NO: 14 to SEQ ID NO: 24;

[0021] The nucleotide sequences of the VP1 genes of PV1, PV2 and PV3 in the poliovirus are shown in SEQ ID NO:25 to SEQ ID NO:27.

[0022] The present invention provides a human enterovirus C group serotype identification primer, including a forward primer EVCY17 with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer EVCZ17 with a nucleotide sequence as shown in SEQ ID NO: 2. The primer provided by the present invention can quickly and accurately detect 23 serotypes of the EV-C group (i.e., Coxsackievirus A, CVA) 1, 11, 13, 17, 19-22, 24, EVC 95, EVC 96, EVC 99, EVC 102, EVC104, EVC 105, EVC 109, EVC 113, EVC116-118 and poliovirus PV1-3), with strong specificity and high sensitivity. It is simpler, faster and more accurate to identify the enterovirus C group serotype, and has good detection operation convenience. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the result of the comparison of enterovirus group C PV1 serotype;

[0024] Figure 2 This is the result of the comparison of enterovirus group C PV3 serotype;

[0025] Figure 3 This is the result of the comparison of enterovirus group C PV2 serotype;

[0026] Figure 4 This is the result of the comparison of enterovirus group C CVA1 serotype;

[0027] Figure 5 This is the result of the comparison of enterovirus group C CVA17 serotype;

[0028] Figure 6 This is the result of the comparison of enterovirus group C CVA22 serotype;

[0029] Figure 7 This is the result of the comparison of enterovirus group C CVA11 serotype;

[0030] Figure 8 This is the result of the comparison of enterovirus group C CVA13 serotype;

[0031] Fig. 9 This is the result of the comparison of enterovirus group C CVA24 serotype;

[0032] Fig.10 This is the result of the comparison of enterovirus group C CVA19 serotype;

[0033] Fig.11 This is the result of the comparison of enterovirus group C CVA20 serotype;

[0034] Fig.12This is the result of the comparison of enterovirus group C CVA21 serotype;

[0035] Fig.13 This is the result of the comparison of enterovirus group C EVC95 serotype;

[0036] Fig.14 This is the result of the comparison of enterovirus group C EVC96 serotype;

[0037] Fig.15 This is the result of the comparison of enterovirus group C EVC99 serotype;

[0038] Fig.16 This is the result of the comparison of enterovirus group C EVC102 serotype;

[0039] Fig.17 This is the result of the comparison of enterovirus group C EVC104 serotype;

[0040] Fig.18 This is the result of the comparison of enterovirus group C EVC105 serotype;

[0041] Fig.19 This is the result of the comparison of enterovirus group C EVC109 serotype;

[0042] Fig. 20 This is the result of the comparison of enterovirus group C EVC113 serotype;

[0043] Fig.21 This is the result of the comparison of enterovirus group C EVC116 serotype;

[0044] Fig. 22 This is the result of the comparison of enterovirus group C EVC117 serotype;

[0045] Fig.23 This is the result of the comparison of enterovirus group C EVC118 serotype;

[0046] Fig.24 The results of detecting enterovirus group A (EV-A) and enterovirus group C (EV-C) positive samples using the primer pairs protected by the present invention; wherein lanes 1 to 8 are EVA71, CVA6, CVA16, CVA10, PV1, PV2, PV3, EVC96, and Marker, respectively;

[0047] Fig.25 To detect the specific results using the primer pairs protected by the present invention, lanes 1 to 9 are EVA71, ECHO30, EVD68, human coronavirus 229E, EVC99, EVC96, CVA17, CVA24, and Marker, respectively;

[0048] Fig.26The results of sensitivity testing using the primer pairs protected by the present invention are shown in Figure 1, wherein 16-1 to 16-6 are 10 2 , 10 1 , 10 0 , 10 -1 , 10 -2 , 10 -3 TCID 50 ;

[0049] Fig. 27 The results of the optimization experiment for the primer pair protected by the present invention. DETAILED DESCRIPTION

[0050] The invention provides a human enterovirus group C serotype identification primer, comprising a forward primer EVCY17 having a nucleotide sequence as shown in SEQ ID NO: 1 (GGNACNCAYRTVATHTGGGA) and a reverse primer EVCZ17 having a nucleotide sequence as shown in SEQ ID NO: 2 (GGRTARTANTYRTTDGCYTCCAT).

[0051] In the present invention, the prototype strains of 23 serotypes of enterovirus group C and the representative strains of different genotypes of each serotype were subjected to whole genome sequence determination, and the above sequence was used as a reference to design a pair of degenerate primers (SEQ ID NO: 3 and SEQ ID NO: 4), but the test results showed that the pair of degenerate primers could not identify the enterovirus group C virus prevalent in China, which may be due to different gene subtypes, some nucleotide sites mutated, and could not match the designed primers. The present invention once again conducted an evolutionary analysis of the genotype and gene subtype of the enterovirus group C virus prevalent in China, re-optimized the degenerate primers according to the nucleotide variation sites, and obtained the above primers protected by the present invention.

[0052] The present invention has no special limitation on the source of the primers, and any primer source known in the art can be used. In the embodiment of the present invention, the primers are preferably synthesized by entrusting Beijing Liuhetong Company, the agent of TaKaRa in China.

[0053] The invention provides a serotype identification kit for enterovirus group C, comprising primers for identifying serotypes of human enterovirus group C and RT-PCR detection reagents.

[0054] In the present invention, the RT-PCR detection reagent preferably includes IIIOne-Step RT-PCR System with Taq. The kit preferably also includes a nucleic acid extraction reagent. The nucleic acid extraction reagent preferably includes components of an RNA extraction kit RNeasyMini kit.

[0055] The present invention provides application of the human enterovirus C group serotype identification primer in preparing an enterovirus C group serotype identification kit.

[0056] In the present invention, the enterovirus C group serotypes preferably include coxsackievirus serotypes 1, 11, 13, 17, 19-22, 24, enterovirus C group serotypes 95, 96, 99, 102, 104, 105, 109, 113 and 116-118, and poliovirus PV1, PV2 and PV3.

[0057] The present invention provides a method for identifying enterovirus group C serotypes for non-disease diagnosis purposes, comprising the following steps:

[0058] Extracting total RNA from samples to be tested;

[0059] Using the total RNA as a template, a one-step RT-PCR reaction is performed using the human enterovirus group C serotype identification primers to obtain an amplified product;

[0060] The amplified product is sequenced, and the obtained sequencing result is compared with the VP1 gene of each serotype of enterovirus group C. If the comparison is consistent, it means that the sample to be tested contains the corresponding serotype of enterovirus group C, otherwise it means that the sample to be tested does not contain the corresponding serotype of enterovirus group C.

[0061] The present invention extracts total RNA from a sample to be tested.

[0062] The present invention has no particular limitation on the method for extracting total RNA from the sample to be tested, and any method known in the art for extracting total RNA from the sample can be used. In an embodiment of the present invention, the method for extracting total RNA from the sample to be tested is preferably performed using a viral RNA extraction kit QIAamp Viral RNA Mini Kit (GIAGEN, Germany).

[0063] After obtaining the total RNA, the present invention uses the total RNA as a template and the human enterovirus group C serotype identification primers to perform a one-step RT-PCR reaction to obtain an amplified product.

[0064] In the present invention, the total volume of the amplification reaction system of the one-step RT-PCR reaction is preferably 25 μL, including 12.5 μL of 2×Reaction Mix buffer, 5.5 μL of RNA free H2O, 0.5 μL of EVCY17, 0.5 μL of EVCZ17, 1.0 μL of RT / Platinum Taq Mix enzyme, and 5.0 μL of template RNA. The amplification reaction program of the one-step RT-PCR reaction is preferably 50°C for 30 min; 94°C for 2 min; 94°C for 30 s, 52°C for 40 s, 72°C for 1 min 20 s, for a total of 40 cycles; and 72°C for 7 min.

[0065] In the present invention, after the one-step RT-PCR reaction is completed, agarose gel electrophoresis is preferably performed, and gel excision and recovery are performed according to the amplification results of each sample to be tested in the electrophoresis diagram. In the embodiment of the present invention, the gel excision and recovery are completed with reference to the instructions of the StartPrep Rapid DNA Gel Recovery Kit.

[0066] After obtaining the amplified product, the present invention sequences the amplified product, and compares the obtained sequencing result with the VP1 gene of each serotype of enterovirus group C. If the comparison is consistent, it means that the sample to be tested contains the corresponding serotype of enterovirus group C, otherwise it means that the sample to be tested does not contain the corresponding serotype of enterovirus group C.

[0067] In the present invention, the sequencing is preferably bidirectional sequencing. After bidirectional sequencing, the forward and reverse primer sequencing results are manually spliced ​​to obtain a complete sequencing result. The present invention has no particular restrictions on the sequence alignment method and the software used, and the alignment method and alignment software known in the art can be used.

[0068] In the present invention, the sequences corresponding to the VP1 genes of each serotype in the enterovirus C group are shown in Table 1:

[0069] Table 1 VP1 gene sequences of each serotype in group C enterovirus

[0070] Serotype Nucleotide sequence (sequence number) Coxsackievirus 1 SEQ ID NO:5 Coxsackievirus 11 SEQ ID NO:6 Coxsackievirus 13 SEQ ID NO:7 Coxsackievirus 17 SEQ ID NO:8 Coxsackievirus 19 SEQ ID NO:9 Coxsackievirus 20 SEQ ID NO:10 Coxsackievirus 21 SEQ ID NO:11 Coxsackievirus 22 SEQ ID NO:12 Coxsackievirus 24 SEQ ID NO:13 Enterovirus C 95 SEQ ID NO:14 Enterovirus C group 96 SEQ ID NO:15 Enterovirus C 99 SEQ ID NO:16 Enterovirus C group 102 SEQ ID NO:17 Enterovirus C group 104 SEQ ID NO:18 Enterovirus C group 105 SEQ ID NO:19 Enterovirus C group 109 SEQ ID NO:20 Enterovirus C group 113 SEQ ID NO:21 Enterovirus C group 116 SEQ ID NO:22 Enterovirus C group 117 SEQ ID NO:23 Enterovirus C group 118 SEQ ID NO:24 Poliovirus PV1 SEQ ID NO:25 Poliovirus PV2 SEQ ID NO:26 Poliovirus PV3 SEQ ID NO:27

[0071] The examples of the present invention verify that the pair of primers and the kit provided by the present invention can simultaneously identify 23 serotypes of virus group C, have the advantages of both specific primers and degenerate primers, and have the advantages of high sensitivity and strong specificity.

[0072] The following is a detailed description of the primers, detection kit and application for identifying human enterovirus group C serotype provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0073] Description of abbreviations of the present invention:

[0074] AW1 buffer: elution buffer 1;

[0075] AW2 buffer: elution buffer 2;

[0076] Carrier RNA: carrier RNA;

[0077] AVL: viral lysis buffer;

[0078] AVEBuffer: protein removal washing buffer;

[0079] PrimeScript One Step RT-PCR Kit Ver.2 (TaKaRa, Dalian, China): one-step reverse transcription polymerase chain reaction;

[0080] 2×Reaction Mix buffer: reaction mixture;

[0081] RNA free H2O: RNA-free water;

[0082] RT / Platinum Taq Mix enzyme: thermostable reverse transcriptase;

[0083] Template RNA: template ribonucleic acid;

[0084] DNA: deoxyribonucleic acid;

[0085] 1×TAE: nucleic acid electrophoresis buffer;

[0086] PCR: polymerase chain reaction;

[0087] Marker 2000: marker 2000;

[0088] QG buffer: dissolution and binding buffer;

[0089] MicroAmp Optical 96-well Plate: Microwell reaction optical 96-well plate;

[0090] All reagents, raw materials, etc. used in the specification are known products and can be purchased.

[0091] Example 1

[0092] A serotype identification kit for enterovirus group C

[0093] 1. Composition

[0094] Enterovirus group C serotype identification primers: EVCY17 with nucleic acid sequences as shown in SEQ ID NO: 1 and EVCZ17 as shown in SEQ ID NO: 2;

[0095] RNA extraction kit RNeasyMini kit (QIAGEN, Germany);

[0096] PCR reaction kit ( ⅢOne-Step RT-PCR with Taq).

[0097] 2. Usage

[0098] 1. Nucleic acid extraction

[0099] (1) When using the kit for the first time, the reagents need to be prepared: add anhydrous ethanol to dilute the AW1 and AW2 buffers according to the volume indicated on the reagent bottle; add 310μL AVE buffer to the bottle containing 310μg Carrier RNA dry powder, shake and mix, and centrifuge instantly. Add 310μL of the above mixture to AVL (31mL) to make AVL working solution, mix well and set aside. After all reagents are prepared, mark the bottle caps and indicate the preparation date;

[0100] (2) Take a 1.5 mL centrifuge tube, add 560 μL AVL working solution and 140 μL sample, oscillate and mix for 15 seconds, and place at room temperature for 10 minutes; In this example, VP1 whole genome sequencing and serotype identification were performed for samples S1-S23 (serotype detection results are PV1, PV2, PV3, CVA1, CVA11, CVA13, CVA17, CVA19, CVA20, CVA21, CVA22, CVA24, EV-C95, EV-C96, EV-C99, EV-C102, EV-C104, EV-C105, EV-C109, CV-C113, EV-C116, EV-C117, EV-C118);

[0101] (3) After instant centrifugation, add 560 μL of anhydrous ethanol, shake and mix for 15 seconds, and centrifuge again;

[0102] (4) Pipette 630 μL of the mixed solution into the separation column, centrifuge at 8000 rpm for 1 min, and replace the collection tube;

[0103] (5) Repeat step 4;

[0104] (6) Add 500 μL of AW1 buffer to the separation column, centrifuge at 8000 rpm for 1 min, and replace the collection tube;

[0105] (7) Add 500 μL AW2 buffer to the separation column, centrifuge at 14,000 rpm for 3 min, replace the collection tube, and centrifuge again at full speed at 14,000 rpm for 1 min;

[0106] (8) Place the separation column in a new 1.5 mL centrifuge tube, add 50 μL AVE Buffer, leave at room temperature for 1 minute, then centrifuge at 8000 rpm for 1 minute, discard the separation column, obtain total viral RNA, place in a 4°C refrigerator or on ice for later use, or place in a -70°C refrigerator for long-term storage.

[0107] 2. Target gene amplification

[0108] (1) Take 5 μL of qualified RNA sample (Ct≤35) and transfer it to a sterile, enzyme-free 200 μL PCR tube;

[0109] (2) Prepare PCR Mix according to the system in Table 1 (this step uses a conventional RT-PCR system). ⅢOne-Step RT-PCR with Taq).

[0110] Table 1 PCR reaction system

[0111] name volume 2×RnxMix 12.5μL SSⅢPlatinumTaqMix 1μL EVCY17 0.5μL EVCZ17 0.5μL RNA 5μL <![CDATA[ddH2O]]> Add to 25 μL

[0112] 3. Electrophoresis

[0113] The PCR product was subjected to DNA horizontal electrophoresis using 1.5% agarose gel electrophoresis, and the gel band was cut for gel recovery experiment. For details, please refer to the StartPrep Rapid DNA Gel Recovery Kit.

[0114] 4. Sequencing comparison

[0115] The obtained gel recovery product was sent to the company for sequencing, and the whole genome sequence of the enterovirus VP1 region to be tested was obtained by sequencing. The sequences with poor quality in the sequencing result map were corrected, and finally the complete VP1 region sequence was obtained. The VP1 sequence obtained by sequencing was compared with the VP1 sequence of the standard strain of all serotypes of enterovirus group C using Sequencher 5.4.5 software (GeneCode, Ann Arbor, Michigan, USA). The similarity was set to 75% or above, and a contig could be synthesized to prove that the two belonged to the same serotype. The results are shown in Figure 1 to Figure 23 The present invention utilizes the above primer pairs to obtain accurate enterovirus serotype identification results.

[0116] Example 2

[0117] Detection of enterovirus group A (EV-A) and enterovirus group C (EV-C) positive samples

[0118] 1. Experimental virus strains

[0119] Enterovirus EV-A serotype representative strains EV-A71, CVA6, CVA16, CVA10 (corresponding to Figure 1 1, 2, 3, 4) Enterovirus C serotype representative strains PV1, PV2, PV3, EVC96 (corresponding to Figure 1 5, 6, 7, 8) were provided by our laboratory. At the same time, the virus titer was titrated (TCID 50 / mL), TCID 50 That is, the tissue cell culture half-infectious dose, each group of strains was 1TCID 50 of virus.

[0120] The above specimens were detected using the kit of Example 1.

[0121] 2. Experimental Results

[0122] Agarose gel electrophoresis results Fig.24 As shown. The primers in Example 1 were used to amplify the four virus strains PV1, PV2, PV3, and EVC96 (corresponding to Figure 1 The sequencing results showed that they were PV1, PV2, PV3, and EVC96 viruses. The primers in Example 1 did not amplify the representative EVA strains EV-A71, CVA6, CVA16, and CVA10 (corresponding to Figure 1 1, 2, 3, 4)

[0123] Example 3

[0124] Specificity test

[0125] 1. Experimental virus strains

[0126] Enterovirus EV-A representative strain (EV-A71) (sample number: 1), enterovirus EV-B representative strain (ECHO30) (sample number: 2), enterovirus EVC-D representative strain (EV-D68) (sample number: 3), human coronavirus 229E (sample number: 4), enterovirus EV-C representative strains (EVC99, EVC96, CVA17, CVA24) (sample numbers: 5, 6, 7, 8), the above virus nucleic acids are all provided by our laboratory. At the same time, the virus titer titration (TCID 50 / mL), TCID 50 That is, the tissue cell culture half-infectious dose, each group of strains was 1TCID 50 of virus.

[0127] 2. Experimental Results

[0128] Agarose gel electrophoresis results Fig.25As shown. The VP1 fragments of the two strains of EVC99 and EVC96 were amplified using the primers in Example 1. The sequencing results showed that they were EVC99 and EVC96 viruses. However, several other viruses, EV-A71, ECHO30, EV-D68 and human coronavirus 229E, were not amplified, indicating that the above-mentioned serotype identification primers for enterovirus group C have good specificity.

[0129] Example 4

[0130] Sensitivity test

[0131] 1. Experimental virus strains

[0132] The serotype identification specific primers in Example 1 were used to detect different concentrations of EVC99, and the EVC99 virus was titrated (TCID 50 / mL), TCID 50 That is, the tissue cell culture half-infectious dose, each group of strains were 1×TCID 50 The viral reference strain was diluted to 10 -3 TCID 50 / mL, 10 -2 TCID 50 / mL, 10 -1 TCID 50 / mL, 10 0 TCID 50 / mL, 10 1 TCID 50 / mL, 10 2 TCID 50 There are 6 groups of dilutions / mL, and the primers in Example 1 are used for detection respectively.

[0133] 2. Experimental Results

[0134] Results Fig.26 The results showed that the minimum detection limit of the virus reached 0.1TCID 50 / ml, which shows that the primers for enterovirus group C in Example 1 have good sensitivity.

[0135] Example 5

[0136] Optimization experiment of primers for serotype identification of enterovirus C group

[0137] In order to identify 23 serotypes of enterovirus group C using a pair of primers in one reaction system, the present invention determined the VP1 whole genome sequence of the prototype strains of the 23 serotypes of enterovirus group C and the representative strains of different genotypes of each serotype, and used the above sequence as a reference to design a pair of degenerate primers as follows:

[0138] Forward primer: ATGYTIGGIACICAYITIATHTGGGA (SEQ ID NO: 3)

[0139] Reverse primer: ATRTTRCADATYTTGTAICCIGC (SEQ ID NO: 4).

[0140] Some of the enterovirus group C strains that cannot be amplified in China include CVA24, EVC99, PV3, and EVC96. The amplification reaction system and conditions are the same as those in Example 1.

[0141] The results are as follows Fig. 27 As shown. In the process of testing the above primers, it was found that some enterovirus group C viruses that have been prevalent in China recently could not be identified. We analyzed that although these strains belong to the same serotype and even the same genotype as the reference strains used in the design of the above primers, they belong to different genetic subtypes and some nucleotide sites have mutated and cannot match the primers.

[0142] In order to confirm this speculation, the present invention has carried out evolutionary analysis of genotypes and subtypes of enterovirus group C viruses that are prevalent in China recently, and confirmed the existence of nucleotide variation sites, and these nucleotide variation sites determine the classification of different subtypes. According to these variation sites, SEQ ID NO: 3 and SEQ ID NO: 4 are re-optimized to obtain the primers shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0143] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a primer for identifying the serotype of human enterovirus group C in preparing a kit for identifying the serotype of enterovirus group C, wherein the primer for identifying the serotype of human enterovirus group C comprises a forward primer EVCY17 having a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer EVCZ17 having a nucleotide sequence as shown in SEQ ID NO: 2; The enterovirus group C serotypes consist of the following species: Coxsackievirus serotypes 1, 11, 13, 17, 19-22, 24, enterovirus group C serotypes 95, 96, 99, 102, 104, 105, 109, 113 and 116-118, and poliovirus PV1, PV2 and PV3.

2. The use according to claim 1, characterized in that: The enterovirus C group serotype identification kit comprises IIIOne-Step RT-PCR System with Taq.

3. The application according to claim 1, characterized in that: Also included are nucleic acid extraction reagents.

4. The use according to claim 3, characterized in that: The nucleic acid extraction reagent includes components of an RNA extraction kit RNeasy Mini kit.

5. A method for identifying enterovirus group C serotypes for non-disease diagnosis purposes, characterized in that: The following steps are involved: Extracting total RNA from samples to be tested; Using the total RNA as a template, a one-step RT-PCR reaction is performed using the primers for identifying the human enterovirus group C serotype described in claim 1 to obtain an amplified product; The amplified product is sequenced, and the obtained sequencing result is compared with the VP1 gene of each serotype of enterovirus group C. If the comparison is consistent, it means that the sample to be tested contains the corresponding serotype of enterovirus group C, otherwise it means that the sample to be tested does not contain the corresponding serotype of enterovirus group C; The serotypes of the enterovirus C group are composed of the following species: serotypes 1, 11, 13, 17, 19-22, 24 in Coxsackievirus, serotypes 95, 96, 99, 102, 104, 105, 109, 113 and 116-118 in the enterovirus C group, and PV1, PV2 and PV3 in the poliovirus.

6. The identification method according to claim 5, characterized in that: The total volume of the amplification reaction system of the one-step RT-PCR reaction is 25 μL, including 12.5 μL of 2×Reaction Mix buffer, 5.5 μL of RNA free H2O, 0.5 μL of EVCY17, 0.5 μL of EVCZ17, 1.0 μL of RT / Platinum Taq Mix enzyme, and 5.0 μL of template RNA; The amplification reaction program of the one-step RT-PCR reaction is preferably 50°C for 30 min; 94°C for 2 min; 94°C for 30 s, 52°C for 40 s, 72°C for 1 min 20 s, for a total of 40 cycles; and extension at 72°C for 7 min.

7. The identification method according to claim 5 or 6, characterized in that: The nucleotide sequences of the VP1 genes of serotypes 1, 11, 13, 17, 19 to 22, and 24 of Coxsackievirus are shown in SEQ ID NO: 5 to SEQ ID NO: 13; The nucleotide sequences of the VP1 genes of serotypes 95, 96, 99, 102, 104, 105, 109, 113 and 116 to 118 in the enterovirus C group are shown in SEQ ID NO: 14 to SEQ ID NO: 24; The nucleotide sequences of the VP1 genes of PV1, PV2 and PV3 in the poliovirus are shown in SEQ ID NO:25 to SEQ ID NO:27.

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