Primer group, kit and method for detecting enterovirus 71 type whole genome and application

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a primer group, a kit and a method for detecting an enterovirus 71 type whole genome and application. According to the invention, a set of specific primer group aiming at EV-A71 whole genome sequence amplification is designed, a multiple PCR sequencing method is combined, two rounds of multiple PCR primers are designed, the defect that amplification is incomplete due to high mutability of enteroviruses is made up, the EV-A71 whole sequence is easier to obtain, pathogen capture can be quickly realized at low cost in disease outbreak and epidemic periods, and the primer group has a wide application prospect. And the method has good application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and in particular relates to a primer set, a kit, a method and an application for detecting the whole genome of enterovirus type 71. Background Art

[0002] Human enterovirus is the main pathogen that causes hand, foot and mouth disease in infants and young children. EV-A71 infection may develop into serious neurological diseases such as brainstem encephalitis and aseptic meningitis, and even cause fatal encephalitis in neonates. Epidemics of neurological hand, foot and mouth disease related to EV-A71 have been widely reported. Studies have shown that 80% of laboratory-confirmed severe cases and 93% of deaths are attributed to EV-A71 infection. Different serotypes and genomic characteristics of EV-A71 have an important influence on pathogenicity. Currently, there is no specific treatment for hand, foot and mouth disease, and symptomatic treatment can only be carried out according to the patient's clinical symptoms.

[0003] The Illumina platform is one of the most mature platforms in the second-generation sequencing, and it occupies a dominant position in the field of short-read sequencing. Currently, most medical institutions have a complete platform for high-throughput sequencing. However, due to the high mutation characteristics of EV-A71, there is a lack of efficient second-generation sequencing primers for EV-A71 on the market, and it is difficult to accurately capture the whole genome sequence of the virus. The whole genome sequence capture carried out by sequencing companies is costly and time-consuming. Therefore, the development of a high-throughput sequencing primer set for the EV-A71 virus to quickly identify pathogen typing and obtain the genomic characteristics of the infected virus strain is particularly important for the clinical treatment of patients and the prevention of severe clinical progress. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a primer set, a kit, a method and an application for detecting the whole genome of enterovirus type 71. The present invention designs a primer set specifically for amplifying the whole genome sequence of EV-A71, and combines the multiplex PCR sequencing method to design two rounds of multiplex PCR primers, which makes up for the disadvantage of incomplete amplification due to the high mutability of enterovirus, making it easier for the present invention to obtain the whole sequence of EV-A71. Based on the above research results, the present invention is completed.

[0005] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a primer set for detecting the whole genome of EV-A71 virus, wherein the primer set is used to detect the whole genome sequence of EV-A71 virus. Specifically, the primer set can amplify the complete genome sequence of EV-A71 virus.

[0007] Furthermore, the nucleotide sequences of the primers contained in the primer set are shown in SEQ ID NO.1 to SEQ ID NO.80.

[0008] The second aspect of the present invention is the use of the above primer set in the preparation of a product for determining the EV-A7 strain in hand, foot and mouth disease.

[0009] The third aspect of the present invention provides a kit for detecting the whole genome of EV-A71 virus, wherein the kit at least comprises the above-mentioned primer set.

[0010] A fourth aspect of the present invention provides use of the above primer set or the above kit in constructing an EV-A71 virus library.

[0011] A fifth aspect of the present invention provides a method for detecting the whole genome of EV-A71 virus based on second-generation sequencing, which is used for non-diagnostic purposes and comprises the following steps:

[0012] S1. Extraction and reverse transcription of RNA from EV-A71 virus samples; S2. Design of amplification primers and establishment of primer amplification system and amplification program to amplify the cDNA obtained by reverse transcription in S1; S3. Purification of PCR amplification products and construction of libraries; S4. Computer detection; S5. Data processing; S6. Obtaining the complete gene sequence of EV-A71.

[0013] A fifth aspect of the present invention provides an application of the above-mentioned method for detecting the whole genome of EV-A71 virus based on second-generation sequencing, wherein the application is one or more of the following:

[0014] (a) Obtain the complete gene sequence of EV-A71 viruses of different types; (b) Used to determine the type of hand, foot and mouth disease.

[0015] Beneficial technical effects of one or more of the above technical solutions:

[0016] (1) Based on the Illumina platform and combined with the whole genome information of human enterovirus 71 (EV-A71), this paper designs a new, efficient and inexpensive second-generation sequencing method.

[0017] (2) The present invention combines the multiplex PCR sequencing method and designs two rounds of multiplex PCR primers, which makes up for the disadvantage of incomplete amplification due to the high mutability of enterovirus, making it easier for the present invention to obtain the full sequence of EV-A71.

[0018] (3) The present invention has designed a complete and feasible specific process for efficient second-generation sequencing of EV-A71, and a specific kit can be developed based on the primer design and reference sequence library of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 is a flowchart of the second generation sequencing of EV-A71 of the present invention;

[0021] Figure 2 This is a simulation diagram of the first round of primer amplification of the EV-A71 virus of the present invention;

[0022] Figure 3 This is a simulation diagram of the second round of primer amplification of the EV-A71 virus of the present invention;

[0023] Figure 4 is a sequence mapping diagram of two EV-A71 strains after the first round of primer amplification in Example 1 of the present invention, wherein a is a sequence mapping diagram of EV-A71 strain 1, and b is a sequence mapping diagram of EV-A71 strain 2;

[0024] Figure 5 This is a sequence mapping diagram of EV-A71 strain 2 after the second round of primer amplification in Example 2 of the present invention;

[0025] Figure 6 This is the phylogenetic tree constructed after the EV-A71 strain in the embodiment of the present invention is tested. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0027] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0028] In a typical embodiment of the present invention, a primer set for detecting the whole genome of EV-A71 virus is provided, and the primer set is used to detect the whole genome sequence of EV-A71 virus. Specifically, the present invention refers to the whole genome sequence of EV-A71 and designs 40 pairs of primers that can cover the whole genome and overlap each other head to tail as the first round of primers, and is named 1L, 1R, 2L, 2R...40L, 40R in sequence. The primers numbered as odd numbers belong to primer set A, and the primers numbered as even numbers belong to primer set B. The basis for adopting this pooling strategy is to amplify at intervals to avoid the formation of dimers due to the close distance between primers and the occurrence of non-specific amplification. The primers of the same group can be mixed together for PCR, and different primer sets are amplified in different tubes, and finally the PCR products of A and B tubes are mixed in the same tube for subsequent library construction. The above primer set can amplify the complete sequence within the primer range on the gene coding region, including any type of mutation sequence within the primer range. When the first round of primer sets fail to measure the complete genome sequence, the second round of primer sets are used for supplementary testing. This operation uses two rounds of multiplex PCR to supplement the gene fragments with low concentrations in the first round of amplification to achieve the purpose of measuring the complete sequence.

[0029] Furthermore, the present invention simulates the amplification of the primers used to ensure that the entire amplification can perfectly contain the entire sequence. The simulated amplification results are as follows: Figure 2 and Figure 3 As shown. The first digit of the primer number indicates the primer group, the second digit indicates the number of the amplified fragment, and the letters (F and R) indicate the upstream and downstream primers. For example, in "1-2F", "1" indicates that the primer group is primer group A, "2" indicates that the amplified fragment is numbered 2 (the specific position corresponds to the fragment number in the simulated amplification result.), and "F" indicates the upstream primer. It can be seen from the two simulated amplification result figures that both rounds of primer design can amplify the full sequence of the EV-A71 virus.

[0030] In another specific embodiment of the present invention, the amplified product is approximately between 250-420 bp.

[0031] In another specific embodiment of the present invention, multiple groups of EV-A71 virus full gene sequences are referenced, and the sizes of the amplification overlap regions of adjacent primers are adjusted to ensure that the multiple primers are designed in the conserved region of the gene.

[0032] In another specific embodiment of the present invention, the nucleotide sequences of the primers contained in the first round primer set in the above primer set are shown as SEQ ID NO.1 to SEQ ID NO.80.

[0033] Table 1 Primer sequences of the first round primer set

[0034]

[0035]

[0036]

[0037] In another specific embodiment of the present invention, the nucleotide sequences of the second round primer set for supplementary testing are shown as SEQ ID NO.81 to SEQ ID NO.132.

[0038] Table 2 Primer sequences of the second round primer set

[0039]

[0040]

[0041] In another specific embodiment of the present invention, after a single virus is amplified by a primer set, the amplification products of two primer pools of the same virus can be mixed, and then adapter sequences can be added to different virus strains by experimental means, and then the libraries of different strains can be mixed and homogenized in a quantitative manner. This method can achieve simultaneous detection of multiple samples through different adapters.

[0042] In another specific embodiment of the present invention, there is provided the use of the above primer set in the preparation of a product for determining the EV-A71 strain in hand, foot and mouth disease.

[0043] In another specific embodiment of the present invention, a kit for detecting the whole genome of EV-A71 virus is provided, and the kit at least comprises the above-mentioned primer set.

[0044] In another specific embodiment of the present invention, the kit further comprises reagents for multiplex PCR amplification reaction, wherein the reagents for amplification reaction comprise DNA polymerase, reaction buffer and enhancer, and the enhancer is betaine.

[0045] In another specific embodiment of the present invention, there is provided use of the above primer set or the above kit in constructing an EV-A71 virus library.

[0046] In another specific embodiment of the present invention, a method for detecting the whole genome of EV-A71 virus based on second-generation sequencing is provided, which is used for non-diagnostic purposes and comprises the following steps:

[0047] S1. Extraction and reverse transcription of RNA from EV-A71 virus samples; S2. Design of amplification primers and establishment of primer amplification system and amplification program to amplify the cDNA obtained by reverse transcription in S1; S3. Purification of PCR amplification products and construction of libraries; S4. Computer detection; S5. Data processing; S6. Obtaining the complete gene sequence of EV-A71.

[0048] In another specific embodiment of the present invention, the primer set in step S2 includes primer set A and primer set B, and the nucleotide sequences thereof are shown in SEQ ID NO.1 to SEQ ID NO.80.

[0049] In another specific embodiment of the present invention, the data processing in step S5 includes arranging the spliced ​​sequences of the detected EV-A71 virus samples and screening the EV-A71 virus samples whose full sequences have not been detected, and the EV-A71 virus samples whose full sequences have not been detected are re-detected using primers with nucleotide sequences such as SEQ ID NO.93 to SEQ ID NO.132.

[0050] In another specific embodiment of the present invention, the data processing in step 5 includes: importing the offline data into the CLCGenomics Workbench platform, comparing it with the established reference sequence library, screening out the reference strain corresponding to the strain, downloading the reference sequence according to its genbank number, and performing MAPPING comparison on the virus against the reference sequence.

[0051] In another specific implementation of the present invention, in step S6, the sequence with complete alignment results is exported according to the MAPPING results.

[0052] In another specific embodiment of the present invention, an application of the above-mentioned method for detecting the whole genome of EV-A71 virus based on second-generation sequencing is provided, and the application is one or more of the following:

[0053] (a) Obtain the complete gene sequence of EV-A71 viruses of different types; (b) Used to determine the type of hand, foot and mouth disease.

[0054] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0055] Example 1

[0056] Next-generation sequencing was used to perform whole genome sequencing on two EV-A71 strains that had been identified and successfully isolated in the laboratory earlier.

[0057] (1) First, extract the viral RNA:

[0058] The kit used for extraction was the viral DNA / RNA extraction kit of Tianlong Technology (Cat. No.: T800), and the experimental steps were carried out according to the instructions.

[0059] (2) Reverse transcription of the extracted two viral RNAs:

[0060] Kit used: Takara reverse transcription kit PrimeScript IV 1st strand cDNA SynthesisMix (Cat. No.: 6215A). The reaction system and procedure are shown in Table 3 and Table 4, respectively.

[0061] Table 3 Reverse transcription reaction system

[0062] Reagents Usage 5x PrimeScript IV cDNA Synthesis Mix 4μL Random 6mers (50μM) 1μL Viral RNA template 10μL <![CDATA[RNase Free H2O]]> 5μL

[0063] Table 4 Reaction procedure

[0064] 30℃ 10min 42℃ 15min 70℃ 15min

[0065] (3) The reverse transcription products of each virus strain were divided into tubes A and B for PCR amplification, wherein the primers used in tube A were SEQ ID NOs. 1 to 40, and the primers used in tube B were SEQ ID NOs. 41 to 80. The kit used for DNA amplification was Takara PremixTaq TM (Cat. No.: RR003A), the reaction system and procedure are shown in Tables 5 and 6, respectively.

[0066] Table 5 PCR reaction system

[0067]

[0068]

[0069] Table 6 Reaction procedure

[0070]

[0071] (4) Purify PCR amplification products and construct library:

[0072] The kits used were Novozymes' second-generation amplification kit (Cat. No.: ND637-C1) and adapter kit DNA Adapters set3-set6 for Illnmina (Cat. No.: N805 / N806 / N807 / N808)

[0073] DNA fragmentation: The amplified product was fragmented. The reaction system and procedure are shown in Tables 7 and 8.

[0074] Table 7 DNA fragmentation system

[0075] Reagents Usage Input DNA 35μL (17.5μL each from tube A and tube B) FEA Buffer 5μL FEA Enzyme Mix 10μL

[0076] Table 8 DNA fragmentation reaction conditions

[0077] Heating cover 105℃ On 37℃ 10 minutes 65℃ 30min

[0078] Linking sequencing adapters: Add adapter sequences to the above reaction products. The reaction system and procedures are shown in Tables 9 and 10, respectively.

[0079] Table 9 DNA fragment plus adapter system

[0080] Reagents Usage Previous step product 60μL Rapid Ligation Buffer-C2 30μL Rapid DNA Ligase 5μL DNA Adapter X 5μL

[0081] Table 10 DNA fragment plus adapter reaction program

[0082] Heating cover 105℃ On 20℃ 15min 4℃ Hold

[0083] (5) Purify the above reaction products using VAHTSDNA Clean Beads:

[0084] 1) After the magnetic beads are equilibrated to room temperature, vortex and oscillate to mix the VAHTSDNA Clean Beads; 2) Pipette 80μL VAHTSDNA Clean Beads into 100μL Adapter Ligation product, vortex or use a pipette to gently blow 10 times to mix thoroughly; 3) Incubate at room temperature for 5 minutes; 4) Centrifuge the PCR tube briefly and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution is clear (about 5 minutes), carefully remove the supernatant; 5) Keep the PCR tube in the magnetic rack, add 200μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant; 6) Repeat step 5 for a total of two rinses; 7) Keep the PCR tube in the magnetic rack, open the lid and air-dry the magnetic beads for 5-10 minutes until no ethanol remains; 8) Remove the PCR tube from the magnetic rack and elute: add 22.5μL sterile ultrapure water to elute, vortex or use a pipette to gently blow to mix thoroughly, place at room temperature for 2 minutes, centrifuge the PCR tube briefly and place it on a magnetic rack to stand. After the solution is clear (about 5 minutes), carefully transfer 20μL of supernatant to a new EP tube without touching the magnetic beads.

[0085] (6) Library construction and homogenization. The specific amplification reaction system and procedures are shown in Tables 11 and 12.

[0086] Table 11 Library amplification system

[0087]

[0088]

[0089] Table 12 Library amplification reaction program

[0090]

[0091] The purification steps after library amplification are the same as those after adding adapters, except that V (magnetic beads): V (library amplification product) = 0.9:1 in ②, that is, 45μL VAHTSDNA Clean Beads are pipetted into 50μL LibraryAmplification product. After the amplified library is purified using magnetic beads, Qubit measures the concentration of the library corresponding to each strain, and the concentrations of all strain libraries to be tested are normalized and mixed into one tube for detection.

[0092] The concentration of the multi-sample mixture is the concentration after dilution by ten times, and one replicate is made for each virus, as shown in Table 13 below.

[0093] Table 13 Concentration of mixed solution of multiple samples

[0094]

[0095] Mix V1-1, V1-2, V2-1, and V2-2 and re-measure the concentration C0 = 3.58 ng / μL.

[0096] Take V mixed solution = 46.2 / C0 = 12.91 μL from the above mixture and put it into a new EP tube, and add water to 50 μL.

[0097] (7) On-machine testing; the testing instrument is produced by Illumina v2Reagent Kit (300Cycles PE). v2Reageent Kit 300Cycles PE, sequence 50 μL of the above-mentioned homogenized solution according to the instructions.

[0098] (8) Data processing;

[0099] The offline data was imported into the CLC Genomics Workbench platform, and after comparing it with the established reference sequence library, the reference strain corresponding to the strain was screened out, the reference sequence was downloaded according to its genbank number, and the virus was mapped against the reference sequence.

[0100] The final comparison result is as follows Figure 4 As shown, Figure 4 a in the figure is the virus1 sequence mapping diagram. Figure 4 Figure b is the virus2 sequence mapping diagram. It can be seen from the figure that the sequencing results of virus2 are incomplete, so a second round of sequencing can be performed on virus2.

[0101] Example 2

[0102] Virus2 was resequenced using the steps in Example 1. Note that except for step (3), all other steps are the same. In the PCR amplification of step (3), the primers used in tube A are SEQ ID NOs. 81 to 106, and the primers used in tube B are SEQ ID NOs. 107 to 132.

[0103] After processing and comparing the offline data, the results are as follows: Figure 5 As shown in the figure, it can be observed that the full sequence of the viral gene is amplified completely.

[0104] The gene sequence of the strain measured by the present invention was analyzed and found to be EV-A71 virus. Figure 6 Taking the constructed phylogenetic tree as an example, virus1 and virus2 are the complete virus sequences measured by the present invention. It can be seen from the phylogenetic tree that virus1 and virus2 are both EV-A71 strains, and the serotype is C4a.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A primer set for detecting the whole genome of EV-A71 virus, characterized in that: The nucleotide sequences of the primers contained in the primer set are shown as SEQ ID NO.1 to SEQ ID NO.

80.

2. The primer set according to claim 1, characterized in that The primer set also includes primers with nucleotide sequences as shown in SEQ ID NO.81 to SEQ ID NO.

132.

3. Use of the primer set according to any one of claims 1 to 2 in the preparation of a product for determining the EV-A71 strain in hand, foot and mouth disease.

4. A kit for detecting the whole genome of EV-A71 virus, characterized in that: The kit at least comprises the primer set according to any one of claims 1-2.

5. The kit according to claim 5, characterized in that Also included are reagents for multiplex PCR amplification reactions, wherein the reagents for amplification reactions include DNA polymerase, a reaction buffer, and an enhancer.

6. Use of the primer set according to any one of claims 1-2 or the kit according to any one of claims 4-5 in constructing an EV-A71 virus library.

7. A method for detecting the whole genome of EV-A71 virus based on second-generation sequencing, which is used for non-diagnostic purposes, characterized in that: The following steps are involved: S1. Extraction and reverse transcription of RNA from EV-A71 virus samples; S2, design amplification primers and establish a primer amplification system and amplification program to amplify the cDNA obtained by reverse transcription in S1; S3, purify the PCR amplification product and construct the library; S4, on-machine testing; S5. Data processing; S6. Obtain the complete gene sequence of EV-A71.

8. The method according to claim 7, characterized in that: The nucleotide sequences of the primers in step S2 are shown in SEQ ID NO.1 to SEQ ID NO.

80.

9. The method according to claim 7, characterized in that: The data processing in step S5 includes arranging the spliced ​​sequences of the detected EV-A71 virus samples and screening the EV-A71 virus samples whose full sequences have not been detected. The EV-A71 virus samples whose full sequences have not been detected are re-detected using primers with nucleotide sequences such as SEQ ID NO.93 to SEQ ID NO.

132.

10. The use of the method for detecting the whole genome of EV-A71 virus based on second generation sequencing according to any one of claims 7 to 9, characterized in that: The application is one or more of the following: (a) Obtaining the complete gene sequence of EV-A71 viruses of different types; (b) Used to determine the type of hand, foot and mouth disease.

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