Rift valley fever virus whole genome targeted amplification primer pair and application thereof
By designing primer pairs for targeted amplification of the entire Rift Valley Fever virus genome, efficient amplification and monitoring of the entire RVFV genome were achieved, solving the problems of insufficient detection sensitivity and coverage in existing technologies, and providing a detection solution with high sensitivity and wide applicability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- TECH CENT OF GUANGZHOU CUSTOMS
- Filing Date
- 2025-02-18
- Publication Date
- 2026-07-21
AI Technical Summary
Existing technologies are insufficient to achieve high sensitivity and specificity in detecting the entire genome of Rift Valley fever virus, thus failing to meet the needs of real-time monitoring and epidemiological research.
Primer pairs for targeted amplification of the entire Rift Valley fever virus genome were designed. Two rounds of PCR amplification were performed, and DNB libraries were prepared for sequencing and bioinformatics analysis, enabling effective amplification and monitoring of the entire RVFV genome.
It improves the sensitivity and accuracy of detection, can identify different variants, is applicable to a variety of sample types, ensures amplification of multiple target areas, has a wide coverage, is suitable for serum, tissue and mosquito samples, and provides scientific evidence for rapid diagnosis and epidemic tracking.
Smart Images
Figure CN119824143B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of molecular biology and virus detection technology, specifically a primer pair and kit for targeted amplification of the whole genome of Rift Valley fever virus and its application in rapid diagnosis and monitoring. Background Technology
[0002] Rift Valley fever virus (RVFV) is a single-stranded RNA virus primarily transmitted by mosquitoes and other vectors. Belonging to the Bunyaviridae family, its infection not only severely impacts livestock but also poses a potential threat to human health. Human infection with RVFV mainly manifests as flu-like symptoms such as fever, headache, and muscle and joint pain, accompanied by nausea and vomiting. Severe cases can lead to liver damage, bleeding, and death. Therefore, RVFV causes significant economic losses to agricultural production and constitutes a serious public health threat.
[0003] Currently, traditional detection methods for RVFV, including enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR, while capable of detecting the virus to some extent, are mostly limited to the analysis of specific gene fragments. This limitation restricts a comprehensive understanding of viral variants and their transmission routes, failing to meet the needs of real-time monitoring. Meanwhile, monitoring based on whole-genome information can more comprehensively capture viral genetic variations, thus providing more reliable data support for epidemiological studies. Therefore, the development of a highly sensitive and specific Rift Valley fever virus whole-genome targeted amplification kit is particularly urgent. Summary of the Invention
[0004] The purpose of this invention is to provide a Rift Valley Fever Virus whole genome targeted amplification primer pair, which can effectively amplify specific gene regions of Rift Valley Fever Virus to achieve efficient and accurate detection and monitoring of RVFV.
[0005] This invention aims to design primers for the entire RVFV genome to achieve efficient amplification and monitoring of the RVFV genome, providing a scientific basis for rapid diagnosis, epidemic tracking, and the formulation of prevention and control measures. Furthermore, the use of whole-genome data will enable a deeper understanding of the evolutionary dynamics of RVFV, promoting the optimization and implementation of public health policies.
[0006] The first objective of this invention is to provide a primer pair for targeted amplification of the entire genome of Rift Valley fever virus, as shown in Table 11.
[0007] A second objective of this invention is to provide a kit for amplifying the whole genome of Rift Valley fever virus, which contains the aforementioned primer pairs.
[0008] The present invention also provides a method for amplifying the whole genome of Rift Valley fever virus, which involves amplifying the genome of Rift Valley fever virus using the above-mentioned primer pairs, then preparing a DNB library for sequencing and bioinformatics analysis.
[0009] Preferably, the PCR amplification includes two rounds of PCR, and the reaction system for the first round of PCR is:
[0010]
[0011] The second round of PCR system is:
[0012]
[0013] Preferably, the PCR amplification includes two rounds of PCR, and the reaction procedure for the first round of PCR is as follows:
[0014]
[0015]
[0016] The reaction procedure for the second round of PCR is as follows:
[0017]
[0018] This invention also provides the application of the above primer pairs or kits in the detection of the whole genome of Rift Valley fever virus.
[0019] This invention has the following innovations and advantages compared to existing technologies:
[0020] (1) High sensitivity: By designing primers for the whole RVFV genome, different variants can be effectively identified, improving the detection rate and accuracy.
[0021] (2) Diverse applications: It is suitable for a variety of sample types, including serum, tissue and mosquito samples, and has good adaptability.
[0022] (3) Wide coverage: Unlike traditional methods that usually target only specific virus strains or gene fragments, this method can amplify multiple different target regions at the same time, ensuring that multiple RVFV variants can be detected. Attached image description:
[0023] Figure 1 This is a high-throughput sequencing coverage map of RVFV in different segments;
[0024] Figure 2 This is a sequencing coverage diagram of the RVFV Ct35.37 sample. Detailed Implementation
[0025] The following embodiments are further illustrations of the present invention, but not limitations thereof.
[0026] Example 1:
[0027] 1. Nucleic acid extraction: Take RVFV culture medium and extract viral nucleic acid RNA using QIAamp Viral RNA Mini Kit (column extraction method). RT-qPCR is used to detect viral nucleic acid Ct values between 28 and 35 for targeted sequencing.
[0028] 2. Reverse transcription: Prepare the reverse transcription reaction solution, vortex to mix 3 times, 3 seconds each time. Take 10 μL of RNA sample into a new 0.2 mL PCR tube, and pipette 10 μL of reverse transcription reaction solution into each sample tube. Mix by pipetting 10 times, centrifuge briefly, and then perform reverse transcription PCR. The reverse transcription reaction system is shown in Table 1.
[0029] Table 1. Preparation system of reverse transcription reaction solution
[0030]
[0031] The reverse transcription PCR reaction procedure is shown in Table 2.
[0032] Table 2 Reverse Transcription PCR Reaction Procedure
[0033]
[0034]
[0035] 3. First Round PCR: Prepare the first round PCR reaction solution as shown in Table 3. Transfer 10 μL of the reverse transcribed cDNA product to a new 0.2 mL PCR tube. Use a pipette to add 15 μL of the prepared first round PCR reaction solution to the PCR tube. Vortex three times for 3 seconds each time, and then briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the PCR tube on a PCR instrument and proceed with the reaction according to the reaction procedure in Table 4.
[0036] Table 3. Preparation system of the first round of PCR reaction solution
[0037]
[0038] Table 4. First-round PCR reaction procedure
[0039]
[0040] 4. Purification of the first-round PCR product: Mix the DNA Clean Beads thoroughly. Add 32.5 μL of DNA Clean Beads to the first-round PCR product. Gently pipette at least 10 times until all magnetic beads are suspended. For the last pipette cycle, ensure all liquid and magnetic beads are transferred into the tube. Incubate at room temperature for 5 minutes. Briefly centrifuge the PCR tube and then place it on a magnetic rack for 3 minutes until the liquid is clear. Carefully aspirate and discard the supernatant. Keep the PCR tube fixed on the magnetic rack, add 160 μL of 80% ethanol to rinse the magnetic beads and tube walls. Gently pipette 3 times and let stand for 30 seconds. Carefully discard the supernatant. Repeat the washing process once. Aspirate as much liquid as possible from the tube. If a small amount remains on the tube wall, briefly centrifuge the PCR tube, separate it on a magnetic rack, and then aspirate the liquid from the bottom of the tube with a small-range pipette. Keep the PCR tube fixed on the magnetic rack, open the cap, and allow it to dry at room temperature until the surface of the magnetic beads is no longer reflective and cracked. Remove the PCR tube from the magnetic rack, add 6.5 μL of TE buffer to elute DNA, and gently pipette at least 10 times until all magnetic beads are resuspended. Incubate at room temperature for 5 minutes. Briefly centrifuge the PCR tube and set aside.
[0041] 5. Second round PCR: Add 4 μL of PCR Barcode Primer Mix to the sample tube (the purified product from the first round, no magnetic bead adsorption or supernatant transfer is required). Prepare PCR reaction solution II according to the required number of reactions, vortex to mix, and briefly centrifuge. Add 14.5 μL of PCR reaction solution II to each sample tube, vortex to mix 3 times, 3 seconds each time, and briefly centrifuge to collect the liquid at the bottom of the tube. Place the PCR tubes on the PCR instrument and perform the reaction according to the reaction procedure in Table 6. After the reaction is complete, briefly centrifuge the PCR tubes, and the product is then purified in the PCR tubes for the next step.
[0042] Table 5. Preparation system of the second round of PCR reaction solution
[0043]
[0044] Table 6. Second Round PCR Reaction Procedure
[0045]
[0046]
[0047] 6. Purification of Second-Round PCR Products: Mix the DNA Clean Beads thoroughly. Add 25 μL of DNA Clean Beads to the second-round PCR products. Gently pipette at least 10 times until all magnetic beads are suspended. For the last pipette cycle, ensure all liquid and magnetic beads are transferred into the tube. Incubate at room temperature for 5 minutes. Briefly centrifuge the PCR tube and then place it on a magnetic rack for 3 minutes until the liquid is clear. Carefully aspirate and discard the supernatant. Keep the PCR tube fixed on the magnetic rack, add 160 μL of 80% ethanol to rinse the magnetic beads and tube walls. Gently pipette 3 times and let stand for 30 seconds. Carefully discard the supernatant. Repeat the washing process once. Aspirate as much liquid as possible from the tube. If a small amount remains on the tube wall, briefly centrifuge the PCR tube, separate it on a magnetic rack, and then aspirate the liquid from the bottom of the tube with a small-range pipette. Keep the PCR tube fixed on the magnetic rack, open the cap, and allow it to dry at room temperature until the surface of the magnetic beads is no longer reflective and cracked. Remove the PCR tube from the magnetic rack, add 25 μL of TE buffer to elute DNA, and gently pipette at least 10 times until all magnetic beads are suspended. Incubate at room temperature for 5 min. Place the PCR tube back on the magnetic rack and let it stand for 3 min until the liquid is clear. Transfer 23 μL of the supernatant to a new 0.2 mL PCR tube.
[0048] 7. PCR Product Quality Analysis: The purified products from the second round of PCR were quantified using the dsDNA HS Assay Kit. A final PCR product concentration ≥1 ng / μL met the library mixing requirements.
[0049] 8. Library pooling and DNB preparation: After the PCR products pass quantitative quality control, a one-step method is used to prepare DNB reagent. The library is pooled at the required 1:1 volume ratio, with a total mass of 50 ng and a total volume ≤ 20 μL, to ensure at least one DNB preparation. The specific strategy for preparing mixed library DNB is described below.
[0050] DNB pretreatment system and reaction procedure: Take 50 ng of the library, in a volume of 20 μL. Make up any shortfall with TE buffer. Configure the pretreatment system and reaction procedure according to Tables 7 and 8.
[0051] Table 7. Pre-reaction system for DNB preparation
[0052]
[0053] Table 8. Pre-reaction procedures for DNB preparation
[0054]
[0055] DNB Reaction System and Procedure: DNB polymerase mixture II needs to be added rapidly to the pre-reaction products. Prepare the pre-reaction system and procedure according to Tables 9 and 10.
[0056] Table 9. DNB Preparation Reaction System
[0057]
[0058] Table 10 DNB Preparation Reaction Procedure
[0059]
[0060] After DNB preparation, quickly add 20 μL of DNB stop buffer to the reaction product. Slowly add the buffer dropwise using a wide-mouth pipette tip, mixing 5–8 times. Do not shake or vigorous pipetting. Use the Qubit ssDNA Assay Kit and Qubit 4.0 instrument to determine the concentration. A concentration of 8 ng / μL or higher is considered acceptable and can be used for instrumental analysis.
[0061] 9. Dual-label library sequencing: DNB libraries were sequenced using dual-bardcode sequencing with the DNBSEQ-G99RS sequencing kit G99 FCL PE150. To prepare the DNB loading system, use a wide-mouth pipette tip to aspirate 21 μL of DNB + 7 μL of loading buffer II + 1 μL of polymerase mixture II (LC). Mix slowly with the wide-mouth pipette tip, then insert 15 μL of the DNB loading system into the chip. Let stand for 2 minutes before sequencing.
[0062] 10. Bioinformatics Analysis: The Viral Consensus Genomes module was run on the open-source cloud analysis platform CZID (https: / / czid.org / ). The raw fastaq file was uploaded, and the Minimap2 pipeline was used to align with the reference genome. Trim galore was used to filter and quality control the uploaded data. Minimap2 was used to further align the filtered data. The iVar package was used to remove the amplicon primers. Finally, the coverage and sequencing depth of the raw data alignment with the reference genome sequence were detected.
[0063] 11. Primer pairs for targeted amplification of the entire genome of Rift Valley fever virus
[0064] The Rift Valley fever virus whole-genome targeted amplification primer set provided by this invention comprises 103 pairs of primer sequences, as shown in Table 11. The average GC value percentage is 45.25%, the average amplicon length is 180.43 bp, and the genome coverage reaches 98.2%.
[0065] 12. Results of two rounds of PCR amplification product concentration detection
[0066] After the first round of RT-PCR amplification and enrichment, the purified sample was quantitatively analyzed using Qubit, and the results showed that the concentration ranged from 3.32 ng / μL to 108 ng / μL. A purified concentration greater than 1 ng / μL was sufficient for subsequent library construction. After a second round of RT-PCR amplification and enrichment, quantitative detection showed that the product concentration ranged from 1.03 ng / μL to 16.73 ng / μL, meeting the standards for sequencing (see Table 12). By designing RVFV-specific primers for amplification and purification, efficient enrichment of the target product was achieved.
[0067] 13. Results of average coverage depth and coverage area tests
[0068] The obtained sequencing data underwent bioinformatics analysis. Sequence fragments were aligned to the L (KX611605.1), M (KX611606.1), S1 (KX611607.1), and S2 (KX611607.1) segments of the RVFV genome, and the average coverage depth and coverage width of each segment were calculated. The results are shown in Table 13 and... Figure 1 As shown, the Ct values ranged from 28 to 35. All samples achieved over 99% coverage across different genomic fragments, with consistent amplification efficiency. Comparison with reference strains revealed average coverage depths between 10215.2x and 225923.8x. While a standard sequencing depth of 100x is generally accepted, the sequencing results from this patent demonstrate extremely high coverage and depth, ensuring comprehensive capture of the target region and guaranteeing data authenticity.
[0069] 14. Sequencing coverage detection results of RVFV samples at high Ct values (Ct35)
[0070] When the Ct value is 35.37, the coverage of the L, M, and S genomes is 99.94%, 99.70%, and 100%, respectively, and the detection limit of this reagent is Ct35. The genome coverage of L, M, and S in high Ct value (Ct35) RVFV samples is shown below. Figure 2 As shown.
[0071] 15. Application of Rift Valley Fever Virus Whole Genome Targeted Amplification Primers in Detecting Unknown Rift Valley Fever Virus Samples
[0072] Sequencing analysis was performed on viral samples collected in our laboratory using Rift Valley fever virus whole-genome targeted amplification primers. These samples were from an unknown virus sample obtained through a UN sequencing technology proficiency testing program, and underwent three freeze-thaw cycles before being stored at -70°C for three months. The results (Table 14) show that, compared to metagenomic sequencing results, amplification and library construction using the primers of this invention effectively increased the number of reads in the target genome, significantly improving sequencing depth and genome coverage. This indicates that the primers remain highly applicable even with poor sample quality.
[0073] Table 11 Primer pairs for RVFV whole-genome targeted amplification
[0074]
[0075]
[0076]
[0077]
[0078]
[0079]
[0080]
[0081]
[0082] Table 12 Qubit quantification of purified products from two PCR experiments
[0083]
[0084] Table 13 RVFV L, M and S genome fragment coverage at different Ct values
[0085]
[0086]
[0087] Table 14 Application of Rift Valley Fever Virus Whole Genome Targeting Amplification Primers in Unknown Virus Samples
[0088]
[0089]
Claims
1. A primer pair for targeted amplification of the entire genome of Rift Valley fever virus, characterized in that, The details are as follows: 。 2. A kit for amplifying the whole genome of Rift Valley fever virus, characterized in that, Contains the primer pair as described in claim 1.
3. A method for amplifying the whole genome of Rift Valley fever virus for non-disease diagnostic and therapeutic purposes, characterized in that, The genome of Rift Valley fever virus was amplified using the primer pair described in claim 1, and then a DNB library was prepared for sequencing and bioinformatics analysis.
4. The method according to claim 3, characterized in that, The amplification includes two rounds of PCR. The reaction system for the first round of PCR is: The second round of PCR system is: 。 5. The method according to claim 3, characterized in that, The amplification involves two rounds of PCR. The reaction procedure for the first round of PCR is as follows: The reaction procedure for the second round of PCR is as follows: 。 6. The use of the primer pair of claim 1 or the kit of claim 2 in the detection of the whole genome of Rift Valley fever virus for non-disease diagnosis and treatment purposes.