Rift valley fever virus whole genome targeted amplification primer pair and application thereof

Abstract

The invention discloses a rift valley fever virus whole genome targeted amplification primer pair and application thereof. The invention aims to realize effective amplification and monitoring of the RVFV whole genome by designing the RVFV whole genome primer, and provides a scientific basis for rapid diagnosis, epidemic situation tracking and formulation of prevention and control measures. In addition, by means of whole genome data, the evolution dynamics of RVFV can be understood more deeply, and optimization and implementation of public health policies are promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and virus detection, and specifically is a primer pair and a kit for targeted amplification of the whole genome of Rift Valley fever virus and the application thereof in rapid diagnosis and monitoring. Background Art

[0002] Rift Valley fever virus (RVFV) is a single-stranded RNA virus that is mainly transmitted by vectors such as Aedes mosquitoes. It belongs to the Bunyaviridae family. Its infection not only has a serious impact on livestock, but also poses a potential threat to human health. Human infection with RVFV mainly manifests as flu-like symptoms such as fever, headache, muscle and joint pain, accompanied by nausea and vomiting. Severe cases can cause liver damage, bleeding and death. Therefore, RVFV not only causes huge economic losses to agricultural production, but also poses a serious public health threat.

[0003] At present, traditional detection methods for RVFV, including enzyme-linked immunosorbent assay (ELISA) and fluorescent quantitative PCR, can detect the virus to a certain extent, but are mostly limited to the analysis of specific gene fragments. This limitation restricts the comprehensive understanding of viral variants and their transmission routes, and cannot meet the needs of real-time monitoring. At the same time, monitoring based on whole genome information can more comprehensively capture the genetic variation of the virus, thereby providing more reliable data support for epidemiological studies. Therefore, it is particularly urgent to develop a highly sensitive and specific Rift Valley fever virus whole genome targeted amplification kit. Summary of the invention

[0004] The purpose of the present invention is to provide a Rift Valley fever virus whole genome targeted amplification primer pair, which can effectively amplify the specific gene region of the Rift Valley fever virus to achieve efficient and accurate detection and monitoring of RVFV.

[0005] The present invention aims to achieve effective amplification and monitoring of the RVFV genome by designing RVFV genome primers, providing a scientific basis for rapid diagnosis, epidemic tracking, and the formulation of prevention and control measures. In addition, with the help of whole genome data, we will be able to have a deeper understanding of the evolutionary dynamics of RVFV and promote the optimization and implementation of public health policies.

[0006] The first object of the present invention is to provide a primer pair for targeted amplification of the whole genome of Rift Valley fever virus, as shown in Table 11.

[0007] The second object of the present invention is to provide a kit for amplifying the whole genome of Rift Valley fever virus, which contains the above-mentioned primer pair.

[0008] The present invention also provides a method for amplifying the whole genome of Rift Valley fever virus, which comprises amplifying the genome of Rift Valley fever virus with the above primer pair, and then preparing a DNB library for sequencing and bioinformatics analysis.

[0009] Preferably, the PCR amplification includes 2 rounds of PCR, and the reaction system of the first round of PCR is:

[0010]

[0011] The second round of PCR system is:

[0012]

[0013] Preferably, the PCR amplification comprises 2 rounds of PCR, and the reaction procedure of the first round of PCR is:

[0014]

[0015]

[0016] The reaction procedure for the second round of PCR is:

[0017]

[0018] The present invention also provides the use of the primer pair or the kit in detecting the whole genome of Rift Valley fever virus.

[0019] Compared with the prior art, the present invention has the following innovations and advantages:

[0020] (1) High sensitivity: By designing primers targeting the entire RVFV genome, different variants can be effectively identified, thereby 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 only target specific virus strains or gene fragments, this approach can simultaneously amplify multiple different target regions, ensuring that multiple RVFV variants can be detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the coverage diagram of high-throughput sequencing of RVFV in different segments;

[0024] Figure 2 This is the sequencing coverage map of the RVFV Ct35.37 sample. DETAILED DESCRIPTION

[0025] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0026] Embodiment 1:

[0027] 1. Nucleic acid extraction: RVFV culture medium was taken, and viral nucleic acid RNA was extracted using QIAamp Viral RNA Mini Kit (column extraction method). RT-qPCR was used to detect viral nucleic acid samples with Ct values ​​between 28 and 35 for targeted sequencing.

[0028] 2. Reverse transcription: prepare reverse transcription reaction solution, vortex mix 3 times, 3 seconds each time, take 10μL RNA sample into a new 0.2mL PCR tube, use a pipette to draw 10μL reverse transcription reaction solution into each sample tube, pipette 10 times to mix, centrifuge for a moment, and then perform reverse transcription PCR. The reverse transcription reaction system is shown in Table 1.

[0029] Table 1 Reverse transcription reaction solution preparation system

[0030]

[0031] The reverse transcription PCR reaction program 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. The reaction system is shown in Table 3. Take 10 μL of the reverse transcribed cDNA product to a new 0.2 mL PCR tube, use a pipette to draw 15 μL of the prepared first round PCR reaction solution and add it to the PCR tube. Vortex 3 times, 3 seconds each time, and centrifuge for a short time to collect the reaction solution to the bottom of the tube. Place the PCR tube on the PCR instrument and react according to the reaction program in Table 4.

[0036] Table 3 Preparation system of the first round PCR reaction solution

[0037]

[0038] Table 4 First round PCR reaction program

[0039]

[0040] 4. Purification of the first round of PCR products: Mix DNA Clean Beads, pipette 32.5μL DNA Clean Beads into the first round of PCR products, and gently blow at least 10 times with a pipette until all the magnetic beads are suspended. The last time, make sure that all the liquid and magnetic beads in the pipette tip are pumped into the tube. Incubate at room temperature for 5 minutes. Centrifuge the PCR tube instantly, then place it on a magnetic rack and let it stand for 3 minutes until the liquid is clear. Use a pipette to carefully pipette the supernatant and discard it. Keep the PCR tube fixed on the magnetic rack, add 160μL 80% ethanol to rinse the magnetic beads and the tube wall, gently blow 3 times, let it stand for 30 seconds, and carefully discard the supernatant. Repeat the washing once. Try to dry the liquid in the tube. If there is a small amount of residue on the tube wall, centrifuge the PCR tube instantly. After separation on the magnetic rack, use a small-range pipette to dry the liquid at the bottom of the tube. Keep the PCR tube fixed on the magnetic rack, open the tube cover, and dry at room temperature until the surface of the magnetic beads is not reflective or cracked. Remove the PCR tube from the magnetic stand, add 6.5 μL TE Buffer to elute the DNA, and gently pipette at least 10 times until all the magnetic beads are suspended. Incubate at room temperature for 5 minutes. Centrifuge the PCR tube briefly and set aside.

[0041] 5. Second round of PCR: Pipette 4μL PCR Barcode Primer Mix into the sample tube (the first round of purified product, no need for magnetic bead adsorption and transfer of supernatant). Prepare PCR reaction solution II according to the required number of reactions, vortex to mix, and centrifuge instantly. Pipette 14.5μL PCR reaction solution II into each sample tube, vortex to mix 3 times, 3s each time, and centrifuge instantly to collect the liquid to the bottom of the tube. Place the PCR tube on the PCR instrument and react according to the reaction program in Table 6. After the reaction is completed, centrifuge the PCR tube instantly and the product will be purified in the PCR tube for the next step.

[0042] Table 5 Preparation system of the second round PCR reaction solution

[0043]

[0044] Table 6 Second round PCR reaction program

[0045]

[0046]

[0047] 6. Purification of the second round of PCR products: Mix the DNA Clean Beads, pipette 25μL DNA Clean Beads into the second round of PCR products, and gently blow at least 10 times with a pipette until all the magnetic beads are suspended. The last time, make sure that all the liquid and magnetic beads in the pipette tip are pumped into the tube. Incubate at room temperature for 5 minutes. Centrifuge the PCR tube instantly, then place it on the magnetic rack and let it stand for 3 minutes until the liquid is clear. Use a pipette to carefully aspirate the supernatant and discard it. Keep the PCR tube fixed on the magnetic rack, add 160μL 80% ethanol to rinse the magnetic beads and the tube wall, gently blow 3 times, let it stand for 30 seconds, and carefully aspirate the supernatant. Repeat the washing once. Try to dry the liquid in the tube. If there is a small amount of residue on the tube wall, centrifuge the PCR tube instantly. After separation on the magnetic rack, use a small-range pipette to absorb the liquid at the bottom of the tube. Keep the PCR tube fixed on the magnetic rack, open the tube cover, and dry it at room temperature until the surface of the magnetic beads is not reflective or cracked. Remove the PCR tube from the magnetic stand, add 25 μL TE Buffer to elute the DNA, and gently pipette at least 10 times until all the magnetic beads are suspended. Incubate at room temperature for 5 minutes. Place the PCR tube on the magnetic stand and let it stand for 3 minutes until the liquid is clear. Transfer 23 μL of the supernatant to a new 0.2 mL PCR tube.

[0048] 7. PCR product quality inspection: Use dsDNA HS Assay Kit to quantify the second round of PCR purification products. The final PCR product concentration ≥1ng / μL meets the library mixing requirements.

[0049] 8. Library pooling and DNB preparation: After the PCR product is qualified in quantitative quality inspection, a one-step method is selected to prepare DNB reagents. The library is pooled at the required 1:1 volume. The total mass after mixing is 50ng, and the total volume is ≤20μL, which satisfies at least 1 DNB preparation. The preparation strategy of the mixed library DNB is described as follows.

[0050] DNB pre-reaction system and reaction procedure: Take 50 ng of library and make the volume 20 μL. Use TE Buffer to make up the insufficient volume. Configure the pre-reaction system and reaction procedure according to Table 7 and Table 8.

[0051] Table 7DNB preparation pre-reaction system

[0052]

[0053] Table 8DNB preparation pre-reaction program

[0054]

[0055] DNB reaction system and reaction procedure: DNB polymerase mixture II needs to be added to the pre-reaction product quickly. Prepare the pre-reaction system and reaction procedure according to Table 9 and Table 10.

[0056] Table 9 DNB preparation reaction system

[0057]

[0058] Table 10DNB preparation reaction procedure

[0059]

[0060] After DNB preparation is completed, quickly add 20 μL of DNB stop buffer to the reaction product, slowly pipette and add dropwise to mix 5 to 8 times with a wide-mouth pipette tip, and do not shake or blow violently. Use Qubit ssDNA Assay Kit and Qubit 4.0 instrument to test the concentration. The concentration of 8 ng / μL or above is qualified, and the instrument can be operated.

[0061] 9. Dual-label library sequencing: DNB library was subjected to dual Bardcode sequencing with DNBSEQ-G99RS sequencing kit G99 FCL PE150 set. Prepare the DNB loading system, use a wide-mouth pipette tip to absorb 21μL DNB+7μL loading buffer II+1μL polymerase mixture II (LC), slowly mix with the wide-mouth pipette tip, absorb 15μL DNB loading system into the chip, let it stand for 2 minutes and then load it on the machine.

[0062] 10. Bioinformatics analysis: Run the Viral Consensus Genomes module through the open source cloud analysis platform CZID (https: / / czid.org / ), upload the original fastaq file, and run the pipeline Minimap2 to align the reference genome. Trim galore filters and quality controls the uploaded data. Minimap2 further aligns the filtered data, and the iVar package removes the amplicon primers. Finally, the coverage and sequencing depth of the original data compared with the reference genome sequence are detected.

[0063] 11. Primer pairs for targeted amplification of the whole genome of Rift Valley fever virus

[0064] The primer combination for targeted amplification of the whole genome of Rift Valley fever virus provided by the present invention comprises 103 pairs of primer sequences, and the primer sequences are shown in Table 11. The average percentage of GC value is 45.25%, the average length of amplicon is 180.43 bp, and the genome coverage reaches 98.2%.

[0065] 12. Results of concentration test of two-round PCR amplification products

[0066] After the first round of RT-PCR amplification and enrichment, the purified samples were subjected to Qubit quantitative analysis, and the results showed that the concentration range was between 3.32ng / μL and 108ng / μL. The concentration after purification only needs to be greater than 1ng / μL to meet the needs of subsequent library construction. After the second round of RT-PCR amplification and enrichment, the quantitative detection results showed that the product concentration was between 1.03ng / μL and 16.73ng / μL, which met the standards for sequencing (see Table 12). By designing RVFV-specific primers, amplifying and purifying the products, efficient enrichment of the target products was achieved.

[0067] 13. Average coverage depth and coverage breadth test results

[0068] The obtained sequencing data were subjected to bioinformatics analysis, and the sequencing fragment sequences 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 breadth of each segment site were calculated. The results are shown in Table 13 and Figure 1 As shown, the Ct value range is 28 to 35. The coverage of all samples in different genomic fragments exceeds 99%, and the amplification efficiency remains uniform. Through comparison analysis with reference strains, the average coverage depth is between 10215.2x and 225923.8x. Generally, the sequencing depth standard is 100x. The sequencing results of this patent show extremely high coverage and depth, ensuring comprehensive capture of the target area and ensuring the authenticity of the data.

[0069] 14. RVFV sample sequencing coverage test results at high Ct value (Ct35)

[0070] When the Ct value is 35.37, the coverage of the L, M and S whole genomes is 99.94%, 99.70% and 100% respectively, and the detection limit of the reagent is Ct35. The whole genome coverage of L, M and S of the high Ct value (Ct35) RVFV sample is shown in the figure. Figure 2 shown.

[0071] 15. Application of Rift Valley Fever Virus Whole Genome Targeted Amplification Primers in Detecting Unknown Rift Valley Fever Virus Samples

[0072] The virus samples collected in this laboratory were sequenced and analyzed using Rift Valley fever virus whole genome targeted amplification primers. The sample was derived from an unknown virus sample in the United Nations sequencing technology capability verification. It was frozen and thawed three times and stored in a -70°C ultra-low temperature refrigerator for three months. The results show (Table 14) that compared with the metagenomic sequencing results, the use of the primers of the present invention for amplification and library construction can effectively increase the number of reads of the target genome, significantly improve the sequencing depth and genome coverage. This shows that even in the case of poor sample quality, the primers still have good applicability.

[0073] Table 11 RVFV whole genome targeted amplification primer pairs

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] Table 12 Qubit quantification of the two PCR purified products

[0083]

[0084] Table 13 Coverage of RVFV L, M and S genome fragments at different Ct values

[0085]

[0086]

[0087] Table 14 Application of Rift Valley fever virus whole genome targeted amplification primers in unknown virus samples

[0088]

[0089]

Claims

1. A primer pair for targeted amplification of the whole 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: Containing the primer pair according to claim 1.

3. A method for amplifying the whole genome of Rift Valley fever virus, characterized in that: The primer pair described in claim 1 is used to amplify the genome of the Rift Valley fever virus, and then a DNB library is prepared for sequencing and bioinformatics analysis.

4. The method according to claim 3, characterized in that The PCR amplification includes 2 rounds of PCR, and the reaction system of the first round of PCR is: The second round of PCR system is:

5. The method according to claim 3, characterized in that: The PCR amplification includes 2 rounds of PCR, and the reaction procedure of the first round of PCR is: The reaction procedure for the second round of PCR is:

6. Use of the primer pair according to claim 1 or the kit according to claim 2 in detecting the whole genome of Rift Valley fever virus.

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