Primer, kit and method for molecular traceability and variation analysis of dengue virus type I

Abstract

The invention relates to the technical field of virus detection, in particular to a primer, a kit and a method for molecular traceability and variation analysis of dengue virus type I. The primer comprises an upstream primer and a downstream primer, the upstream primer and the downstream primer are both subjected to Tag and locked nucleic acid dual modification; the upstream primer has a nucleotide sequence as shown in SEQ ID NO 15; and the downstream primer has an amino acid sequence as shown in SEQ ID NO 16. The complete E gene sequence is obtained through sequencing after the complete E gene is subjected to RT-PCR amplification, time and labor are saved, the cost is low, and the complete E gene can be completely analyzed. The invention aims to establish an amplification primer capable of amplifying a complete E gene of the dengue virus type I (Den1) and a kit comprising the amplification primer, an amplification reagent and the like, sequencing an amplification product, and further performing molecular traceability and virus variation analysis on the virus.

Description

Technical Field

[0001] The present application relates to the technical field of virus detection, and in particular to a primer and a kit, and a method for molecular tracing and variation analysis of dengue virus type I. Background Art

[0002] Dengue virus (DENV) belongs to a serotype subgroup within the genus Flavivirus in the family Flaviviridae, which includes a variety of single-stranded positive-sense RNA viruses. In addition to dengue virus, this genus also includes West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV).

[0003] Dengue virus is morphologically similar to Japanese encephalitis virus (JE) but is smaller, measuring approximately 17-25 nm. It is primarily transmitted by vector insects such as Aedes aegypti and Aedes albopictus, causing dengue fever, as well as dengue hemorrhagic fever and dengue shock syndrome, which are associated with significant morbidity and mortality.

[0004] Dengue virus particles are spherical, with a diameter of 45-55 nm. There are four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), all of which can cause severe infection in humans. DENV-4 is less transmissible and has a smaller reach. Currently, DENV-1 and DENV-2 are the most prevalent serotypes of dengue virus. The genome is a single-stranded, positive-sense RNA containing a single open reading frame, which encodes three structural proteins and seven non-structural proteins.

[0005] Typically, suspected dengue virus infection cases are initially characterized using methods such as dengue virus antigen, nucleic acid, or antibody testing to provide a preliminary diagnosis of dengue virus infection. Fluorescence quantitative PCR is then used for preliminary quantification and serotype determination (DENV-1, DENV-2, DENV-3, and DENV-4). Based on the serotype and quantitative results (copy number determined by the ct value and standard curve), specific primers that amplify the complete E gene are selected for amplification. The amplified product is sequenced to obtain a sequence, and the virus is then molecularly traced and analyzed for mutations based on the sequence.

[0006] Currently, numerous molecular tracing and variant analysis methods exist. One option is to directly sequence the entire dengue virus genome before conducting molecular tracing and variant analysis. This approach is the most comprehensive and accurate, but it is expensive and time-consuming, making it unsuitable for widespread adoption. Another option is to amplify the core variant region of the E gene and then use next-generation sequencing to obtain the sequence for tracing and variant analysis. This method is inexpensive and time-saving, but because it only analyzes the core variant region, other regions of the E gene cannot be analyzed, potentially resulting in inaccurate tracing and variant analysis.

[0007] Therefore, it is necessary to establish a new method for molecular tracing and mutation analysis of dengue virus. Summary of the Invention

[0008] The present application provides a primer and a kit, as well as a method for molecular tracing and variation analysis of dengue virus type I.

[0009] This application chooses to amplify the complete E gene by RT-PCR followed by sequencing to obtain the complete E gene sequence. This method is both time-saving and labor-saving, low-cost, and allows for complete analysis of the entire E gene. The goal is to develop amplification primers and a kit including primers and reagents that can amplify the complete E gene of dengue virus type 1 (Den1), sequence the amplified product, and further perform molecular tracing and viral mutation analysis.

[0010] In a first aspect, the present application provides a primer for amplifying the complete E gene of dengue virus type 1, using the following technical solution:

[0011] A primer for amplifying the complete E gene of dengue virus type I, comprising an upstream primer and a downstream primer; both the upstream primer and the downstream primer are double-modified with a tag and a locked nucleic acid;

[0012] The upstream primer has a nucleotide sequence as shown in SEQ ID NO 15; the Tag sequence used for modification in the upstream primer has a nucleotide sequence as shown in SEQ ID NO 31; the T base of the 25th nucleotide of the upstream primer is modified with a locked nucleic acid;

[0013] The downstream primer is the amino acid sequence shown in SEQ ID NO 16; in the downstream primer, the Tag sequence used for modification is the amino acid sequence shown in SEQ ID NO 32; and the T base of the 27th nucleotide of the downstream primer is modified with a locked nucleic acid.

[0014] In a second aspect, the present application provides a kit for amplifying the complete E gene of dengue virus type I, which includes the above-mentioned primers.

[0015] Optionally, the kit further comprises an enzyme mixture, an amplification buffer, a positive quality control product and a negative quality control product.

[0016] Optionally, the enzyme mixture includes reverse transcriptase, RNase inhibitor, Taq DNA polymerase and enzyme buffer.

[0017] Optionally, the amplification buffer comprises a buffer, dNTPs, Mg 2+ .

[0018] Among them, positive quality control products and negative quality control products are used for quality control during the use of the kit.

[0019] Optionally, all components in the kit can be completely mixed to form a fully premixed form.

[0020] Aliquot the premixed reagents into 8-tube strips and cap them to create a pre-packed format. During use, simply open the tube cap, add the extracted nucleic acid, and cap the tube. This reduces the number of steps and the chance of error.

[0021] Optionally, the kit further comprises sequencing primers for sequencing the sample to be tested.

[0022] The present application selects the conserved regions upstream and downstream of the complete E gene sequence in the Den1 genome sequence and designs upstream and downstream primers respectively. After analyzing the possible dimers, secondary structures, etc. upstream and downstream, appropriate sequences are selected to form amplification primers that can amplify the complete E gene of dengue virus type 1 (Den1). Virus mutations are constantly occurring, resulting in large genomic sequence variations. Traditional primers have certain problems. For example, due to certain mutations of the virus, when designing primers, if the variability of the existing sequence is not reconsidered or the latest mutation is fully considered, the primer compatibility problem will result.

[0023] The present application increases the binding force of the primers to the template by using a Tag sequence at the 5 ends of the upstream and downstream primers (this sequence will not bind to any position in the dengue virus genome) and performing locked nucleic acid modification in the upstream and downstream sequences. Therefore, even if there are some mutations, the template can be amplified well, thereby increasing the success rate of sample amplification.

[0024] The primers or a kit containing the primers are used for molecular tracing and variation analysis of dengue virus. The primers or a kit containing the primers are time-saving, labor-saving, low-cost, and can fully analyze the entire E gene, making them an optimal solution for tracing and variation analysis.

[0025] The principle of using the above primers to amplify the complete E gene of Den1 in this application is as follows:

[0026] Both the upstream primer and the downstream primer in the primer set can specifically bind to the Den1 genomic template. During RT-PCR, if Den1 is present in the sample to be tested, the upstream primer and the downstream primer will bind to the Den1 genomic template. As the RT-PCR proceeds, with each cycle, the upstream and downstream primers will amplify using the product of the previous cycle as a template, when there is sufficient enzyme or other raw materials and no accumulated inhibitors. The product of each amplification is 2 to the power of the number of templates in the previous cycle system. After 30-40 cycles, sufficient products can be accumulated for subsequent gel electrophoresis detection or Sanger sequencing analysis.

[0027] The upstream and downstream primers provided in this application have strong specificity and will not cause non-specific binding. Therefore, the above primers can be used to specifically amplify Den1 in the sample to be tested and obtain the complete E gene sequence.

[0028] In a third aspect, the present application provides a method for molecular tracing and variation analysis of a sample using the above primers or the above kit, employing the following technical solutions:

[0029] A method for molecular tracing and variation analysis of a sample using the above primers or the above kit, the method specifically comprising the following steps:

[0030] (1) Using a nucleic acid extraction kit to extract nucleic acid from the sample to be tested to obtain viral RNA;

[0031] (2) Add the extracted RNA to eight pre-packed tubes to form a reaction system;

[0032] (3) performing RT-PCR amplification on the reaction system to obtain an amplified product;

[0033] (4) performing gel electrophoresis analysis on the amplified products to obtain amplified bands, and performing Sanger sequencing on the amplified products of the positive bands;

[0034] (5) Through comparison and analysis of related sequences, select reference sequences and construct a phylogenetic tree.

[0035] In some specific embodiments, the concentration of each primer in the reaction system is 0.2-0.25 μmol / L.

[0036] In a specific embodiment, the concentrations of the upstream primer and the downstream primer in the reaction system are the same, and the concentration of each primer is 0.2 μmol / L.

[0037] In the present application, by controlling the concentrations of the upstream and downstream primers and probes in the reaction system within the above range, the method has the best effect of amplifying the complete Den1 genome and the highest reaction efficiency; excessively high or low concentrations of the upstream and downstream primers in the reaction system will reduce the amplification effect.

[0038] In some specific embodiments, in step (2), the reaction system is 20 μL, specifically: 6 μL of RNA solution extracted from the sample to be tested, 14 μL of pre-packaged reagents (1 μL of enzyme mixture, 3 μL of primers (including upstream primers and downstream primers), dNTPs and Mg 2+ The buffer was 10 μL).

[0039] In some specific embodiments, in step (3), the amplification conditions include:

[0040] Reverse transcription at 50-55°C for 10-50 min;

[0041] Pre-denaturation at 90-95°C for 1-5 min;

[0042] Denaturation at 90-95°C for 10-50 seconds, annealing at 55-65°C for 10-50 seconds, and extension at 65-75°C for 30-180 seconds, for 30-40 cycles.

[0043] In some preferred embodiments, in step (3), the amplification conditions include:

[0044] Reverse transcription at 50°C for 30 min;

[0045] Pre-denaturation at 94°C for 2 min;

[0046] Denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute were performed for 35 cycles.

[0047] By adopting the above-mentioned amplification conditions, the present application can effectively amplify the complete E gene of Den1 in the sample to be tested.

[0048] In some specific embodiments, in step (4), performing gel electrophoresis analysis and Sanger sequencing on the amplified product includes the following aspects:

[0049] After the test sample is amplified using the kit, the amplified product is subjected to 1.5% agarose gel electrophoresis. First, analyze the positive and negative controls. The positive control should have an amplified band at 1623 bp, while the negative control should have no amplified band. If the amplified band is 1623 bp, the amplification is successful and subsequent Sanger sequencing can be performed.

[0050] Sanger sequencing was performed in both directions using the following sequencing primers:

[0051] Tag-F:AGGTGACACTATAGAATA.

[0052] Tag-R: GTACGACTCACTATAGGGA.

[0053] The obtained sequence was obtained by checking the sequencing peak graph to obtain the accurate sequence. Figure 11 The peaks are the exact sequences. Figure 12 There are double peaks, the sequence is inaccurate, and this sequence needs to be discarded.

[0054] In some specific embodiments, in step (5), constructing a phylogenetic tree and performing phylogenetic tree analysis on the sample sequence includes the following aspects:

[0055] The complete Den1 genome and E gene sequences were downloaded from GenBank. Den1 virus reference sequences from different time periods and regions were screened based on temporal and spatial distribution. Using MEGA11 software, the reference sequences were aligned with the laboratory-derived sequences, and a phylogenetic tree was constructed using the neighbor-joining method. Phylogenetic analysis of viral variation was performed to trace the origin of the virus.

[0056] It is worth noting that the method described in this application is a method for non-disease diagnosis purposes.

[0057] In summary, this application includes at least one of the following beneficial technical effects:

[0058] (1) The origin tracing and variant analysis of dengue virus type I requires whole genome sequencing or complete E gene sequencing, and then the sequence is obtained to conduct origin tracing and variant analysis based on the sequence. Currently, no solution has been found for amplifying the complete E gene for origin tracing and variant analysis.

[0059] The present application selects the conserved regions upstream and downstream of the complete E gene sequence in the Den1 genome sequence and designs upstream and downstream primers respectively. After analyzing the possible dimers, secondary structures, etc. upstream and downstream, appropriate sequences are selected to form amplification primers that can amplify the complete E gene of dengue virus type 1 (Den1). Virus mutations are constantly occurring, resulting in large genomic sequence variations. Traditional primers have certain problems. For example, due to certain mutations of the virus, when designing primers, if the variability of the existing sequence is not reconsidered or the latest mutation is fully considered, the primer compatibility problem will result.

[0060] The present application increases the binding force of the primers to the template by using a Tag sequence at the 5 ends of the upstream and downstream primers (this sequence will not bind to any position in the dengue virus genome) and performing locked nucleic acid modification in the upstream and downstream sequences. Therefore, even if there are some mutations, the template can be amplified well, thereby increasing the success rate of sample amplification.

[0061] (2) The kit can completely mix the above primers, enzyme mixture, amplification buffer, etc. together to form a fully premixed form. The fully premixed reagents are pre-packed into 8 strip tubes and capped to form a pre-packed form. During use, only the tube cap needs to be opened, the extracted nucleic acid is added, and the tube cap is closed before use. This reduces the number of operation steps and the probability of operation errors.

[0062] The method for detecting using the primers provided in this application or a kit including the primers, and the method for amplifying the complete E gene, has the advantages of high sensitivity, simple operation, short detection time, small sample amount required, and low cost. It can directly amplify the nucleic acid extracted from the sample to be tested, and has high application value in virus tracing and variation analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is the amplification result without any modification of the primers in Example 2.

[0064] Figure 2 This is the amplification result of the primers in Example 2 with individual Tag modification.

[0065] Figure 3 This is the amplification result of the primers in Example 2 subjected to individual locked nucleic acid modification.

[0066] Figure 4 This is the amplification result of the primers in Example 2 with double modification of Tag and locked nucleic acid.

[0067] Figure 5 This is the amplification result of the first set of primers in Example 3 in which the primers were double-modified with Tag and locked nucleic acid.

[0068] Figure 6 This is the amplification result of the second set of primers in Example 3 after double modification of the primers with Tag and locked nucleic acid.

[0069] Figure 7 This is the amplification result of the third set of primers in Example 3 after double modification of the primers with Tag and locked nucleic acid.

[0070] Figure 8 These are the amplification results of the samples amplified using the third set of primers that were double-modified with Tag and locked nucleic acid in Example 5 (D1-D6 are the amplification results of samples 1-6, respectively).

[0071] Figure 9 These are the amplification results of the samples amplified using the third set of primers doubly modified with Tag and locked nucleic acid in Example 5 (D7-D11 are the amplification results of samples 7-11, respectively; D12 is the amplification result of the positive control; and D13-D14 are the amplification results of two negative controls).

[0072] Figure 10 This is the phylogenetic tree of the 11 samples in Example 5.

[0073] Figure 11 This is the sequencing peak diagram (accurate sequence).

[0074] Figure 12 This is the sequencing peak diagram (there are double peaks and the sequence is inaccurate).

[0075] Among them, the brightest band in the maker of the gel image is 750bp, and from bottom to top they are 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, and 5000bp. DETAILED DESCRIPTION

[0076] Before describing the embodiments of the present application in detail, it should be understood that the terminology used herein is only for the purpose of describing particular embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the term belongs.

[0077] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of this application, unless otherwise specified, "plurality" means two or more.

[0078] The endpoints of the ranges and any values ​​disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0079] In this application, the term "comprise" or "include" is an open expression, that is, it includes the content specified in this application, but does not exclude other aspects of the content.

[0080] To make the purpose, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without making any creative work are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be understood as limiting this application.

[0081] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0082] The present application is further described in detail below in conjunction with the examples and test results.

[0083] Example 1

[0084] This example provides the design and synthesis of primers.

[0085] A literature search concluded that the E gene is the primary target gene for molecular tracing and variant analysis of dengue virus, and several candidate primer sequences were obtained. The E gene and its upstream and downstream sequences were downloaded from the NCBI nucleotide database. Using the bioinformatics software MEGA-X, a multiple sequence alignment was performed on the upstream and downstream sequences of the dengue virus E gene, identifying multiple conserved segments of the gene that specifically matched dengue virus type 1 and avoided matching other dengue virus strains or other pathogens. Upstream and downstream primers were designed, and structural analysis was performed using PrimerPremier 5 software.

[0086] Finally, primer specificity and compatibility analysis were performed using the NCBI Primer Blast function to ensure that the primer combination was highly specific for amplifying dengue virus type 1 and that no base mutations could lead to missed detection. The specific nucleotide sequences (5'-3') of the three primer combinations ultimately identified for dengue virus type 1 are shown in Table 1.

[0087] Table 1 Three sets of primers

[0088]

[0089] Example 2

[0090] In this example, the first set of primers provided in Example 1 was modified with tags and locked nucleic acids, and their effects on the amplification success rate were explored.

[0091] (1) Modification part

[0092] Taking the first set of primers as an example, the 5' ends of the upstream and downstream primers were modified with either a single tag, a single locked nucleic acid, or both a tag and a locked nucleic acid. An unmodified primer set was used as a control. The modified sequences are shown in Table 2.

[0093] Table 2 Modifications for the first set of sequences

[0094]

[0095] (2) Amplification part

[0096] Use PrimeScript sold by TAKARA TM One Step RT-PCR Kit Ver. 2 (Dye Plus) was used as the amplification reaction system. Ten clinical samples (dengue virus type 1 positive samples) were amplified using the four primer combinations listed in Table 2.

[0097] The reaction system is 20 μL, specifically: 14 μL of pre-packaged reagents, including: 1 μL of enzyme mixture; 10 μL of amplification buffer; 3 μL of primers (the concentrations of upstream primers and downstream primers are the same, both 0.2 μmol / L); 6 μL of extracted RNA solution is directly added to the pre-packaged system.

[0098] The amplification program was set as follows: reverse transcription at 50°C for 30 min; pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 1 min, for 35 cycles.

[0099] After amplification, gel electrophoresis analysis was performed to observe the amplification success rate of the sample and determine the amplification effect of different modifications. The expected size of the amplified product of the four different modified primer combinations was 1623 bp.

[0100] (3) Amplification results

[0101] The amplification results of different modification methods are as follows Figure 1-4 As shown. Among them, Figure 1-4 These are the amplification results of primers without any modification, with single Tag modification, single LNA modification, and dual Tag and LNA modification.

[0102] The amplification of the four modifications was analyzed by gel electrophoresis. Figure 1 It can be seen that without any modification of the amplification primers, 8 out of 10 samples can be amplified successfully; Figure 2 It can be seen that only the Tag-modified amplification primers can successfully amplify 8 out of 10 samples; Figure 3It can be seen that the amplification primers modified with locked nucleic acid alone can successfully amplify 9 out of 10 samples; the amplification primers modified with both tag and locked nucleic acid can successfully amplify all 10 samples.

[0103] Therefore, it can be proved that double modification of Tag and locked nucleic acid has better amplification effect.

[0104] Example 3

[0105] In this example, the second and third sets of primers provided in Example 1 were double-modified with tags and locked nucleic acids, and the amplification performance of the three sets of double-modified primers was explored.

[0106] (1) Modification part

[0107] The second and third sets of primers were double-modified with tags and locked nucleic acids, using the same modification method as described in the "Modification" section of Example 2. The three sets of double-modified primers are shown in Table 3.

[0108] Table 3 Three sets of double-modified primers

[0109]

[0110] (2) Amplification part

[0111] Use PrimeScript sold by TAKARA TM One Step RT-PCR Kit Ver. 2 (Dye Plus) was used as the amplification reaction system. Ten clinical samples (dengue virus type I positive samples) were amplified using the three sets of double-modified primers listed in Table 3.

[0112] The reaction system and amplification procedure were the same as those in the "amplification part" of Example 2.

[0113] After amplification, gel electrophoresis analysis was performed to assess the amplification success rate and confirm the amplification effectiveness of the three sets of doubly modified primers. The expected size of the amplified product for the first primer combination was 1623 bp, the second primer combination was 1574 bp, and the third primer combination was 1583 bp.

[0114] (3) Amplification results

[0115] The amplification results of the three sets of double-modified primers are as follows Figure 5-7 As shown. Among them, Figure 5-7 These are the amplification results of the first, second, and third sets of primers that were double-modified with Tag and locked nucleic acid, respectively.

[0116] The amplification results of the three sets of double-modified amplification primers were analyzed by gel electrophoresis. Figure 5 The amplification results of the first set of amplification primers shown in the figure show that 9 out of 10 samples can be amplified successfully. Figure 6 The amplification results of the second set of amplification primers shown in the figure show that 9 out of 10 samples can be amplified successfully. Figure 7 The amplification results of the third set of amplification primers are shown, and all 10 samples can be amplified successfully.

[0117] Therefore, it can be concluded that compared with the first two sets of primers, the third set of primers has a better amplification effect after double modification. Next, the double-modified third set of primers will be used as the preferred primer combination in the kit.

[0118] Example 4

[0119] This embodiment provides a kit for molecular tracing of dengue virus type 1 (Den1) and virus mutation analysis and amplification. The kit includes the third set of primers in Example 3 that have been double-modified with a tag and locked nucleic acid.

[0120] The above kit specifically includes the following components:

[0121] Component 1: Eight tube strips containing pre-aliquoted amplification reagents (including enzyme mix, amplification buffer, and doubly modified primers).

[0122] Component 2: Sequencing primer composition (including forward and reverse sequencing primers);

[0123] Component 3: positive quality control;

[0124] Component 4: Negative control.

[0125] Example 5

[0126] This example is the application of the kit provided in Example 4.

[0127] The sample was analyzed using the above kit. The specific process is as follows:

[0128] (1) Remove the eight-tube strip containing the pre-packaged amplification reagent from the kit, thaw and add 6 μL of dengue virus type I RNA solution. A total of 11 clinical samples, 2 negative quality control products, and 1 positive quality control product were amplified.

[0129] (2) The amplification program was set as follows: reverse transcription at 50°C for 30 min; pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 1 min, for 35 cycles.

[0130] (3) After amplification, perform gel electrophoresis analysis to observe the amplification of the quality control sample to determine whether the experiment is successful and the amplification of the sample. The expected size of the amplified product is 1583 bp.

[0131] (4) Amplification results

[0132] The amplification results of 11 clinical samples, 2 negative quality control products and 1 positive quality control product are as follows Figure 8-9 Among them, D1-D11 are the amplified bands of 11 samples, D12 is the amplified band of the positive quality control, and D13 and D14 are the amplified bands of the negative quality control.

[0133] The amplification results of the above samples were analyzed by gel electrophoresis. Figure 8-Figure 9 As can be seen, the positive control sample amplified a band of the expected size, while the negative control sample did not amplify, indicating that the control samples amplified normally. 11 samples were fully amplified successfully.

[0134] (5) Sequencing analysis

[0135] Eleven samples were sent for Sanger sequencing. The sequences listed in Table 4 were used as sequencing primers for bidirectional sequencing.

[0136] Table 4 Sequencing primers

[0137] sequence name Sequence (5'-3') SEQ ID NO Tag F AGGTGACACTATAGAATA 17 Tag R GTACGACTCACTATAGGGA 18

[0138] The sequencing results of the E gene of the 11 samples are shown in Table 5 .

[0139] Table 5 Sequencing results of E gene in 11 samples

[0140] Serial number Sample No. SEQ ID NO 1 D1 19 2 D2 20 3 D3 21 4 D4 22 5 D5 23 6 D6 24 7 D7 25 8 D8 26 9 D9 27 10 D10 28 11 D11 29 12 Positive quality control 30

[0141] All relevant sequences were downloaded from Genbank and compared. Based on the temporal and spatial distribution, viruses from different time and regions were selected as reference sequences, and a phylogenetic tree was constructed. The 11 sample sequences were subjected to phylogenetic tree analysis. Figure 10 shown.

[0142] from Figure 10 The specific distribution of the 11 samples in the tree can be seen, which enables the molecular tracing of dengue virus type 1 (Den1) and virus mutation analysis.

[0143] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A primer for amplifying the complete E gene of dengue virus type I, characterized in that: The primers include an upstream primer and a downstream primer; both the upstream primer and the downstream primer are double-modified with a tag and a locked nucleic acid; The upstream primer has a nucleotide sequence as shown in SEQ ID NO 15; the Tag sequence used for modification in the upstream primer has a nucleotide sequence as shown in SEQ ID NO 31; the T base of the 25th nucleotide of the upstream primer is modified with a locked nucleic acid; The downstream primer is the amino acid sequence shown in SEQ ID NO 16; in the downstream primer, the Tag sequence used for modification is the amino acid sequence shown in SEQ ID NO 32; and the T base of the 27th nucleotide of the downstream primer is modified with a locked nucleic acid.

2. A kit for amplifying the complete E gene of dengue virus type I, characterized in that: The kit comprises the primers according to claim 1.

3. The kit according to claim 1, wherein The kit also includes an enzyme mixture, an amplification buffer, a positive quality control product, and a negative quality control product.

4. The kit according to claim 3, wherein The enzyme mixture includes reverse transcriptase, RNase inhibitor, Taq DNA polymerase and enzyme buffer.

5. The kit according to claim 3, characterized in that The amplification buffer comprises a buffer, dNTPs, Mg 2 + .

6. The kit according to claim 3, characterized in that Each component in the kit can be completely mixed to form a fully premixed form.

7. The kit according to claim 3, wherein The kit also includes sequencing primers for sequencing the sample to be tested.

8. A method for molecular tracing and variation analysis of a sample using the primers according to claim 1 or the kit according to any one of claims 2 to 7, characterized in that: The method specifically comprises the following steps: (1) Use a nucleic acid extraction kit to extract nucleic acid from the sample to be tested and obtain viral RNA; (2) Add the extracted RNA to the eight-tube strip containing pre-packed reagents to form a reaction system; (3) performing RT-PCR amplification on the reaction system to obtain an amplified product; (4) performing gel electrophoresis analysis on the amplified product to obtain amplified bands, and performing Sanger sequencing on the amplified product of the positive band; (5) Through comparison and analysis of related sequences, select reference sequences and construct a phylogenetic tree.

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