Multiple amplification primer group capable of obtaining chikungunya virus whole genome through amplification and application of multiple amplification primer group

Abstract

The invention provides a multiple amplification primer group capable of obtaining a chikungunya virus whole genome through amplification and application of the multiple amplification primer group, and belongs to the technical field of biology. The invention provides a multiple amplification primer group capable of obtaining a chikungunya virus whole genome through amplification. The nucleotide sequence of the multiple amplification primer group is shown as SEQ ID NO.1-SEQ ID NO.64. The multiple amplification primer group can obtain the chikungunya virus whole genome through amplification. The multiple amplification primer group provided by the invention covers 99.74% of a Chikungunya virus genome, can completely and uniformly cover the virus genome, and realizes an average coverage depth gt in nanopore sequencing and high-throughput sequencing; and the power is 104 times. Compared with the prior art, the multiple amplification primer group provided by the invention has the advantages that the detection sensitivity and the genome coverage degree are obviously improved, and all sample genomes obtained by amplification are not broken in the splicing process.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a multiplex amplification primer set capable of amplifying the complete genome of Chikungunya virus and its applications. Background Technology

[0002] Chikungunya virus (CHIKV) belongs to the genus *Alphavirus* of the family Occultviridae. CHIKV is transmitted by infected female mosquitoes, most commonly *Aedes aegypti* and *Aedes albopictus*. The virus is mainly distributed in Africa, South Asia, and Southeast Asia. In recent years, with the accelerating pace of globalization, CHIKV has shown a trend of spreading to other regions. With the maturity and cost reduction of sequencing technology, whole-genome sequencing of CHIKV can more accurately genotype and trace the virus's origin. Currently, pathogen-targeted sequencing technology based on multiplex PCR has been applied to the whole-genome capture of various pathogens such as Zika and monkeypox.

[0003] In the previously reported article (Su Wenzhe, Li Yan, Lu Weizhi, et al. (2024), Establishment of a high-throughput sequencing method for the whole genome of Chikungunya virus based on multiplex PCR), the primer pairs for CHIKV amplification length is about 500bp, and there are a total of 113 primers. Using two positive samples from 2025 (Ct values ​​of 19.21 and 25.4, respectively), the primer pool and the amplification conditions in the article were applied for amplification. The results showed that the concentrations of the amplicon after purification were 0.25ng / L and 0.24ng / L, respectively, and the Qsep fragment analysis results did not show the target fragment. The primer pool could not amplify the positive samples from 2025. It can be seen that the existing technology has the following defects: (1) the primer pairs cannot obtain the whole genome of the new pathogen. The published primer pool cannot specifically capture the positive samples of the epidemic in July 2025. Therefore, it is necessary to design a better primer pool for the samples from 2025 in order to obtain the whole genome of CHIKV. (2) The detection sensitivity needs to be improved. In the past, the detection sensitivity required a Ct value ≤ 35, which could not effectively obtain the whole genome for weak positive samples.

[0004] Therefore, in order to better monitor the outbreak, spread and mutation of CHIKV pathogen, it is necessary to develop a scheme that can uniformly capture the entire CHIKV genome for existing sequencing platforms in China. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a multiplex primer set capable of amplifying the whole genome of Chikungunya virus, which significantly improves detection sensitivity and genome coverage compared with existing technologies.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a set of multiplex primers capable of amplifying the complete genome of Chikungunya virus, comprising 32 pairs of primers with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.64.

[0007] This invention also provides the application of the above-mentioned multiplex amplification primer set in the preparation of products for detecting Chikungunya virus.

[0008] Preferably, the product includes reagents or kits.

[0009] The present invention also provides a kit for detecting Chikungunya virus, comprising the above-described multiplex amplification primer set.

[0010] Preferably, the kit includes primer set 1 and primer set 2, wherein primer set 1 includes primer pairs with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.32, and primer set 2 includes primer pairs with nucleotide sequences as shown in SEQ ID NO.33 to SEQ ID NO.64.

[0011] The present invention also provides a method for preparing a sequencing fragment of Chikungunya virus, which uses the above-mentioned multiplex amplification primer set to amplify cDNA obtained by reverse transcription of the sample to be tested, and the amplification product is purified to obtain the sequencing fragment.

[0012] Preferably, primer set 1 and primer set 2 are used to amplify the cDNA obtained by reverse transcription of the sample to be tested, and the amplification products are combined and purified to obtain the sequencing fragment; primer set 1 includes primer pairs with nucleotide sequences as shown in SEQ ID NO.1~SEQ ID NO.32, and primer set 2 includes primer pairs with nucleotide sequences as shown in SEQ ID NO.33~SEQ ID NO.64.

[0013] Preferably, the amplification reaction system, in 12.5 μL increments, consists of 6.25 μL of Q5. ® The mixture consists of Hot Start High-Fidelity 2X Master Mix, 1.75 μL of primer set at a concentration of 10 nM, and 4.5 μL of cDNA.

[0014] Preferably, the amplification reaction program is 98℃ for 30s; 98℃ for 15s, 63℃ for 5min, cycling Ct value + 3 times (the minimum number of cycles is 20, that is, the number of cycles for samples with Ct value ≤ 16 is set to 20); and stored at 4℃.

[0015] Preferably, the sequencing fragment is suitable for second-generation sequencing or third-generation sequencing.

[0016] The beneficial effects of this invention are: The multiplex amplification primer set provided by this invention covers 99.74% of the chikungunya virus genome, achieving complete and uniform coverage of the viral genome. In both nanopore sequencing and high-throughput sequencing, an average coverage depth >10 was achieved. 4 The gene coverage of samples with Ct values ​​≤36.94 reached 100% on both the second-generation sequencing and third-generation sequencing platforms. Samples with Ct values ​​of 38.14 achieved 100% gene coverage on the third-generation sequencing platform, while the gene coverage on the second-generation sequencing platform was as high as 98.88%. Furthermore, all genomes amplified using the multiplex amplification primer set provided by this invention showed no breaks during splicing, and the homology between the second-generation sequencing platform data and the reference genome was as high as 99.96%~100%. In summary, the multiplex amplification primer set provided by this invention significantly improves detection sensitivity and genome coverage compared to existing technologies, providing excellent technical support for obtaining more uniform and effective chikungunya virus genomes. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the coverage of the multiplex amplification primer set of the present invention on the Chikungunya virus. Detailed Implementation

[0018] This invention provides a set of multiplex primers capable of amplifying the complete genome of Chikungunya virus, comprising 32 pairs of primers with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.64.

[0019] This invention comprehensively considers the integrity of RNA in clinical samples and the balance of different amplification products, and finally designed and synthesized 32 pairs of PCR multiplex primers targeting the entire genome of Chikungunya virus. The amplification product of each primer pair is 458nt~473nt. In July 2025, a local outbreak of the epidemic occurred in Foshan, Guangdong, my country, caused by imported cases. According to the epidemiological investigation information, the strain of this outbreak originated from Reunion Island. Therefore, the Chikungunya virus strain 80652-1-1-1 (GenBank number PV700165.1) submitted in May 2025 was used as the reference genome (11426nt). The multiplex primer set designed in this invention covers 99.74% of the Chikungunya virus genome, from 2bp to 11396bp, and can specifically capture the Chikungunya virus strain 80652-1-1-1.

[0020] This invention also provides the application of the above-described multiplex amplification primer set in the preparation of products for detecting Chikungunya virus. In this invention, the products preferably include reagents or kits.

[0021] The present invention also provides a kit for detecting Chikungunya virus, comprising the above-described multiplex amplification primer set.

[0022] In this invention, there is an overlap region of 83nt to 375nt between adjacent primer amplification regions (see schematic diagram of the coverage of the multiplex amplification primer set for Chikungunya virus in this invention). Figure 1 As shown in the figure, the primer sets of this invention are divided into two tubes according to odd and even pairs, referred to as tube 1 and tube 2. Tube 1 contains primer set 1, and tube 2 contains primer set 2, each containing 16 pairs of primers. There are no overlapping regions between primers in the same tube. The primers in tube 1 and tube 2 are named 400bp-Pool 1 and 400bp-Pool 2, respectively. Primer set 1 includes primer pairs with nucleotide sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 32, and primer set 2 includes primer pairs with nucleotide sequences as shown in SEQ ID NO. 33 to SEQ ID NO. 64, as shown in Table 1 below. This invention does not have a specific limitation on the specific preparation method of the primer sequences; conventional primer sequence preparation methods in the art can be used.

[0023] Table 1 Primer sequence information

[0024] The present invention also provides a method for preparing a sequencing fragment of Chikungunya virus, which uses the above-mentioned multiplex amplification primer set to amplify cDNA obtained by reverse transcription of the sample to be tested, and the amplification product is purified to obtain the sequencing fragment.

[0025] In this invention, primer set 1 and primer set 2 are used to amplify cDNA obtained by reverse transcription of the sample to be tested. After merging the amplification products, the sequencing fragment is obtained through purification. Primer set 1 includes primer pairs with nucleotide sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 32, and primer set 2 includes primer pairs with nucleotide sequences as shown in SEQ ID NO. 33 to SEQ ID NO. 64. In this invention, LunaScript is preferably used for reverse transcription. ® cDNA synthesis was performed using the RT SuperMix Kit (NEB, E3010) reverse transcription kit. In this invention, the amplification reaction system is typically 12.5 μL, preferably 6.25 μL of Q5. ®The reaction system consists of Hot Start High-Fidelity 2X Master Mix, 1.75 μL of primer set at a concentration of 10 nM, and 4.5 μL of cDNA. In this invention, the primer set is obtained by mixing primer pairs at a concentration of 10 nM in equal proportions. During amplification, the total amount of primer set added is 1.75 μL. The same sample is amplified using both primer set 1 and primer set 2, with identical reaction systems for both. The preferred amplification reaction program is 98℃ for 30 s; 98℃ for 15 s, 63℃ for 5 min, with a cycle count of Ct value + 3 (the minimum cycle count is 20, i.e., for samples with a Ct value ≤ 16, the cycle count is set to 20); storage at 4℃. The Ct value is the Ct value measured by qRT-PCR, and the amplification cycle count is the same for primer set 1 and primer set 2. The number of cycles used in this invention is not a fixed value, but is dynamically determined based on the Ct value of the sample. The number of cycles = Ct value + 3, meaning the amplification cycle number for each sample is the Ct value measured by qRT-PCR plus 3 cycles (the minimum number of cycles is 20, i.e., the number of cycles for samples with a Ct value ≤ 16 is set to 20). In this invention, the sequencing fragment is suitable for either second-generation sequencing or third-generation sequencing. In this invention, the preferred names of the library preparation kits suitable for second-generation sequencing and third-generation sequencing platforms are: Third-generation sequencing (QPursue-6k): using the Direct Barcode Library Preparation Kit (QNLK-24 V3.0); Second-generation sequencing (DNBSEQ-G99): using the MGIEAasy Fast PCR-FREE Restriction Digestion Library Preparation Kit (940-000884-00).

[0026] In this invention, the number of cycles in the PCR amplification program is dynamically adjusted based on the Ct value of the sample (Ct value + 3, minimum number of cycles 20, i.e., the number of cycles for samples with Ct values ​​≤ 16 is set to 20), optimizing amplification conditions for samples with different viral loads. The primer set provided by this invention is compatible with dual-platform sequencing: a library preparation and sequencing workflow specifically designed for both second-generation sequencing (DNBSEQ-G99) and third-generation sequencing (QPursue-6k) is implemented, achieving platform compatibility. Sensitivity and coverage were validated in this invention: the sensitivity and genome coverage of the amplification system were systematically verified using sequencing results from five samples with different Ct values ​​(17.41~38.14) on both platforms.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Unless otherwise specified, the following embodiments are all conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] Example 1 The multiplex amplification primer sets shown in Table 1 were prepared as follows: Primers were synthesized using the solid-phase phosphoramidite method, purified by PAGE, with a total synthesis volume of 2 OD, and dispensed into 1 OD / tube. After centrifuging the primer powder at 1200 rpm / min for 2 min, water was added according to the molar mass / OD, and nuclease-free water was added to make the concentration of primers in each tube 100 nM. Equal volumes of primers from each tube were mixed to form two primer pools, 400 bp-Pool 1 (primer set 1) and 400 bp-Pool 2 (primer set 2), according to odd and even numbers. The mixed primer pools were then diluted to a working concentration of 10 nM according to the experimental requirements.

[0031] The protocol for amplifying Chikungunya virus-related clinical samples using the aforementioned primer pool is as follows: 1. RNA concentration requirement: The viral RNA obtained from clinical samples must be a positive sample according to the quantitative real-time PCR results.

[0032] 2. Using LunaScript ® cDNA synthesis was performed using the RT SuperMix Kit (NEB, E3010) reverse transcription kit, following the system described in Table 2 and the reaction conditions described in Table 3: Table 2 Reaction preparation system Table 3 Reaction conditions Note: The product of the first strand of reverse transcription can be stored at -20°C. 3. For the same sample, amplify it separately for each pool, using Q5. ® The reaction was carried out using a hot start High-Fidelity 2X Master Mix (NEB, M0494). The reaction system is shown in Table 4, and the reaction procedure is shown in Table 5. Table 4 Multiplex PCR reaction system

[0033] Table 5 Multiplex PCR reaction procedure

[0034] Note: Ct value is the Ct value measured by qRT-PCR. The amplification cycle number is the same for pool 1 and pool 2, and the minimum cycle number is 20. That is, the cycle number is set to 20 for samples with Ct value ≤ 16.

[0035] 4. PCR product purification: Combine the products from both amplification pools into a 1.5 mL EP tube. Purify using 1× volume of Agencourt AMPure XP magnetic beads.

[0036] 5. PCR product quality control: using Qubit ® The dsDNA HS Assay Kit is a double-stranded DNA fluorescence quantitative reagent kit. Follow the instructions of the quantitative kit to quantify the PCR-purified product.

[0037] 6. The fragment distribution of the PCR-purified products was detected using Qsep's electrophoretic separation-based equipment.

[0038] 7. The amplified products can be constructed into libraries according to different sequencing platforms and then subjected to second-generation and third-generation sequencing on the BGI DNBSEQ-G99 and QiCarbon Technology's QPursue-6k nanopore gene sequencer platforms. For specific library construction methods, please refer to the instructions of the corresponding kits.

[0039] Example 2 The multiplex amplification primer set of Example 1 was tested on five samples with different viral loads. Five Chikungunya positive samples with known Ct values ​​were selected, with Ct values ​​of 17.41, 25.19, 30.29, 36.84, and 38.14 respectively (when calculating the number of cycles in the subsequent calculation, each Ct value was calculated as an integer, i.e., the value after the decimal point was discarded).

[0040] Then, after amplifying the samples using the technical solution described in Example 1, the mixed samples of amplified products were library constructed and sequenced using a QPursue-6k nanopore gene sequencer according to the reagent instructions (direct barcode library construction kit (QNLK-24 V3.0, C-006-0030) and sequencing kit (QDS V3.0, C-002-0006)). Simultaneously, these products were also used to construct second-generation sequencing platform libraries (MGIEasy Fast PCR-FREE restriction enzyme digestion library preparation kit (940-000884-00)) according to the reagent instructions, and sequenced using the BGI DNBSEQ-G99 sequencing platform's G99 App-D FCL PE150 sequencing kit (940-001274-00). The sequencing libraries were tagged, allowing for differentiation in the sequencing data. The third-generation sequencing platform generated 3.49 Gbp of sequencing data, and the second-generation sequencing platform generated 8.22 Gbp. Bioinformatics methods are used to align sequencing fragments to a reference genome and calculate the coverage depth of each genomic locus (i.e., how many sequencing fragments cover each locus).

[0041] The sequencing depth coverage of the five samples is shown in Table 6, indicating that the amplification system of this invention achieved relatively uniform coverage of CHIKV. In both nanopore sequencing and high-throughput sequencing, an average coverage depth >10 was achieved. 4 In comparison, the second-generation sequencing platform showed more uniform data yield across samples. Samples with Ct values ​​≤36.94 achieved 100% gene coverage on both platforms, while samples with Ct values ​​38.14 achieved 100% gene coverage on the third-generation sequencing platform and a high gene coverage of 98.88% on the second-generation sequencing platform. Furthermore, all genomes from this invention showed no breaks during assembly, and the data from the second-generation sequencing platform exhibited 99.96%–100% homology with the reference genome.

[0042] Table 6. Sequencing depth coverage of 5 samples

[0043] The above results demonstrate that this invention significantly improves both detection sensitivity and genome coverage, providing a good technical solution for obtaining a more uniform and effective chikungunya virus genome. Specificity is reflected in the following aspects: 1. Sample validation: Amplification and sequencing were performed using five chikungunya virus positive samples with different Ct values, all of which successfully obtained high-coverage genome data. 2. Comparison with existing primers: The published primer pool (113 primers) (Su Wenzhe, Li Yan, Lu Weizhi, et al. (2024), Establishment of a high-throughput sequencing method for the whole genome of chikungunya virus based on multiplex PCR) could not effectively amplify samples from 2025 (concentration only 0.24~0.25 ng / μL), while the primer pool of this invention could effectively amplify them. 3. Dual-platform validation: High coverage was achieved on both second-generation sequencing and third-generation sequencing platforms, indicating that the primer set of this invention has good specificity in different sequencing technologies.

[0044] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A set of multiplex primers capable of amplifying the complete genome of Chikungunya virus, characterized in that, It includes 32 pairs of amplification primers, with nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.

64.

2. The use of the multiplex amplification primer set according to claim 1 in the preparation of products for detecting Chikungunya virus.

3. The application according to claim 2, characterized in that, The products include reagents or kits.

4. A kit for detecting Chikungunya virus, characterized in that, Includes the multiplex amplification primer set as described in claim 1.

5. The reagent kit according to claim 4, characterized in that, The kit includes primer set 1 and primer set 2. Primer set 1 includes primer pairs with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.32, and primer set 2 includes primer pairs with nucleotide sequences as shown in SEQ ID NO.33 to SEQ ID NO.

64.

6. A method for preparing chikungunya virus sequencing fragments, characterized in that, The cDNA obtained by reverse transcription of the sample to be tested was amplified using the multiplex amplification primer set described in claim 1, and the amplification product was purified to obtain the sequencing fragment.

7. The method according to claim 6, characterized in that, cDNA obtained by reverse transcription of the sample to be tested was amplified using primer set 1 and primer set 2 respectively. After merging the amplification products, the sequencing fragment was obtained by purification. Primer set 1 includes primer pairs with nucleotide sequences as shown in SEQ ID NO.1~SEQ ID NO.32, and primer set 2 includes primer pairs with nucleotide sequences as shown in SEQ ID NO.33~SEQ ID NO.

64.

8. The method according to claim 6, characterized in that, The amplification reaction system, in 12.5 μL volume, consists of 6.25 μL of Q5. ® The mixture consists of Hot Start High-Fidelity 2X Master Mix, 1.75 μL of primer set at a concentration of 10 nM, and 4.5 μL of cDNA.

9. The method according to claim 6, characterized in that, The amplification reaction program is 98℃ for 30s; 98℃ for 15s, 63℃ for 5min, cycled Ct value +3 times; stored at 4℃.

10. The method according to claim 6, characterized in that, The sequencing fragments are suitable for second-generation sequencing or third-generation sequencing.

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