A Nipah virus whole genome sequencing kit

Abstract

This invention discloses a Nipah virus whole genome sequencing kit. The kit contains primers shown in SEQ ID NO.1 to SEQ ID NO.189. The primers are used to perform PCR amplification on the samples to be sequenced, followed by enzymatic fragmentation and end repair, adapter ligation, library preparation, and sequencing. The kit is designed to achieve full coverage of both NiV-M and NiV-B genomes. Through a dual-pool shingled primer system, primer dimer formation is effectively blocked, thus ensuring efficient gene fragment amplification and sequencing. In terms of detection sensitivity, this kit can stably detect samples of both genotypes, especially for low-load samples (Ct value > 30), exhibiting high genome coverage and ensuring efficient sample adaptation. It is beneficial for applications in various scenarios such as scientific research, epidemic monitoring, and cross-border quarantine, possessing significant practical value and promising prospects for widespread application.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection and high-throughput sequencing technology, specifically to a Nipah virus whole genome sequencing kit. Background Technology

[0002] Nipah virus (NiV) is a highly pathogenic and infectious single-stranded negative-sense RNA virus belonging to the genus Henipavirus in the family Paramyxoviridae. Its natural host is primarily the fruit bat (Pteropus spp.) of the family Pteropidae. The full-length genome of Nipah virus is approximately 18.2 kb, encoding six major structural and non-structural proteins: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), glycoprotein (G), and polymerase macroprotein (L).

[0003] Based on genomic sequences, Nipah virus is mainly divided into two lineages: the Malaya lineage (NiV-M) and the Bangladeshi lineage (NiV-B). The Bangladeshi lineage has a mortality rate as high as 75%, while the Malaya lineage has a mortality rate of 40%. Transmission routes include direct contact with infected animals, consumption of food contaminated with the bodily fluids, secretions, or excrement of infected animals, and close contact with patients or their bodily fluids. Currently used detection methods include serological methods (ELISA), molecular detection methods (RT-qPCR), and high-throughput sequencing. However, existing molecular diagnostic methods mostly focus on partial gene fragments, resulting in limited detection range, inability to achieve whole-genome coverage and comprehensive typing, and difficulty in meeting the needs of epidemic tracing and evolutionary research. Although metagenomic sequencing (NGS) can provide whole-genome information, it still suffers from shortcomings such as host nucleic acid background interference, operational complexity, poor reproducibility, low detection sensitivity, and difficulty in covering the entire genome. Summary of the Invention

[0004] To address the aforementioned issues, this invention develops a Nipah virus whole-genome sequencing kit based on the Nipah virus genome. This provides a new technology for viral gene detection services and promotes the development of the biopharmaceutical industry.

[0005] The first objective of this invention is to provide primers for whole-genome sequencing of Nipah virus, the sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.189.

[0006] The second objective of this invention is to provide a Nipah virus whole genome sequencing kit containing the aforementioned primers.

[0007] Preferably, the primers are divided into Nipah A Panel Mix and Nipah B Panel Mix, the sequence of Nipah A Panel Mix is ​​shown in SEQ ID NO.1~SEQ ID NO.96, and the sequence of Nipah B Panel Mix is ​​shown in SEQ ID NO.1, SEQ ID NO.97~SEQ ID NO.189.

[0008] Preferably, the kit further includes a reverse transcription system, an amplification system, and a product purification system.

[0009] A third objective of this invention is to provide the application of the aforementioned primers or kits in Nipah virus whole-genome sequencing.

[0010] The fourth objective of this invention is to provide a method for whole-genome sequencing of Nipah virus, which includes the following steps: PCR amplification of the sample to be sequenced using the primers described above, followed by enzymatic fragmentation and end repair, adapter ligation, library preparation, and sequencing.

[0011] Preferably, the PCR amplification system is as follows: tube A: 4 μL Nipah A Panel Mix, 10 μL first-strand cDNA template, 10 μL 3×T Enzyme, and 6 μL Nuclease-free water; tube B: 4 μL Nipah B Panel Mix, 10 μL first-strand cDNA template, 10 μL 3×T Enzyme, and 6 μL Nuclease-free water; the sequence of the Nipah A Panel Mix is ​​shown in SEQ ID NO.1~SEQ ID NO.96, and the sequence of the Nipah B Panel Mix is ​​shown in SEQ ID NO.1, SEQ ID NO.97~SEQ ID NO.189.

[0012] Preferably, the PCR product is purified after the A tube and the B tube are mixed.

[0013] Preferably, the PCR amplification program is: 98℃ for 3 min; 98℃ for 30 sec, 60℃ for 2 min, 72℃ for 1 min, 25 cycles; 72℃ for 1 min; 4℃ Hold.

[0014] Preferably, the sequencing requirements are: 1-5 Million reads, sequencing read length: at least 100bp per end, and data analysis output ≥0.1 Gb / Sample.

[0015] This invention has the following innovations and advantages compared to existing technologies: This invention provides a Nipah virus whole genome sequencing kit to overcome the problems of existing technologies, such as limited detection range to partial fragments, difficulty in achieving comprehensive typing, and limited detection sensitivity. This sequencing kit has good compatibility with both Nipah virus M and B strains, and can effectively obtain the entire Nipah virus genome. It provides excellent data support for virus typing, source tracing analysis, mutation monitoring, and phylogenetic tree construction, and can be widely used in scientific research, monitoring, and source tracing analysis.

[0016] This invention relates to a whole-genome sequencing kit, comprising reverse transcription reagents, multiplex PCR reaction reagents, a Nipah virus primer pool, end-fraction repair reagents, adapter ligation reagents, and other library construction reagents. The primer pool covers the full length of both Nipah virus M and B types, amplifying fragments up to 400 bp in length. The kit is compatible with both Illumina and BGI sequencing platforms for library construction and sequencing. The kit exhibits good detection sensitivity for low-viral-load samples and is suitable for human, animal, and environmental samples with Ct ≤ 35, making it applicable to a wide range of scenarios.

[0017] The kit of this invention is designed to achieve full coverage of the NiV-M and NiV-B genomes. Through a dual-pool tiled primer system, it effectively blocks primer dimer formation, thereby ensuring efficient gene fragment amplification and sequencing. Regarding detection sensitivity, this kit can stably detect samples of both genotypes, especially for low-load samples (Ct value > 30), maintaining high genome coverage and ensuring efficient sample adaptation. With these advantages, this technical solution can be widely applied in various scenarios such as scientific research, epidemic monitoring, and cross-border port quarantine, possessing significant practical value and promising prospects for widespread application. Detailed Implementation

[0018] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0019] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0021] To address the aforementioned technical challenges, this invention designs targeted sequencing primer sets based on the characteristics of the Nipah virus genome and develops whole-genome sequencing kits for Nipah virus strains B and M. For samples with Ct values ​​between 15 and 30, this kit provides 100% genome coverage and sequencing depth typically tens of thousands of times, thus providing crucial technical support for early warning and control of Nipah virus disease outbreaks.

[0022] This invention employs a dual-pool shingled multiplex primer design, dividing the primers for two Nipah virus types into two separate primer pools: NiV-A and NiV-B Panel Mix. This completely eliminates the risk of forming extremely short non-target amplicones (Primerdimers) between overlapping primers, ensuring that target amplicones up to approximately 400 bp can be amplified uniformly and without bias. A total of 189 primer sequences are provided, detailed in Table 9. In the sequences, Y represents C or T, R represents A or G, W represents A or T, S represents G or C, M represents A or C, and K represents G or T.

[0023] Example: I. Sample Pretreatment and Nucleic Acid Extraction 1) Sample pretreatment: Viral RNA was extracted using the silica gel column method with QIAamp Viral RNA Kits (52906). The specific operation followed the instructions of the virus extraction kit.

[0024] 2) Sample quantification method: Real-time quantitative PCR (qPCR) was used to quantify viral RNA.

[0025] II. Database Creation Operation Process Scope of application: This procedure is applicable to Illumina paired-end 150bp (PE150) sequencing mode.

[0026] 1. Reverse transcription Prepare the reverse transcription reaction solution, vortex three times for 3 seconds each time, transfer 10 μL of Nipah virus RNA sample to a new 0.2 mL PCR tube, and pipette 10 μL of reverse transcription reaction solution (excluding RNA nucleic acid) into each sample tube. Mix by pipetting 10 times, briefly centrifuge, and then perform reverse transcription PCR to obtain the first-strand cDNA template. The reverse transcription reaction system is shown in Table 1. The reverse transcription PCR reaction procedure is shown in Table 2. 5× RT Mix was purchased from Hebei Bingyuan Shengkang Medical Technology Co., Ltd., catalog number 12R0170.

[0027] Table 1. Reverse transcription reaction solution preparation system

[0028] Table 2 Reverse Transcription PCR Reaction Procedure

[0029] 2. First round of PCR 2.1 System Configuration and Reaction Prepare the first-round PCR reaction solution as shown in Table 3. Place the PCR tubes on the PCR instrument and proceed with the reaction according to the reaction procedure in Table 4. 3×T Enzyme was purchased from Hebei Bingyuan Shengkang Medical Technology Co., Ltd., catalog number 120003. Nipah A Panel Mix is ​​a mixture of primers from SEQ ID NO.1 to SEQ ID NO.96, and Nipah B Panel Mix is ​​a mixture of primers from SEQ ID NO.1 and SEQ ID NO.97 to SEQ ID NO.189.

[0030] Table 3. Preparation system of the first round of reaction solution

[0031] Table 4. First-round PCR reaction procedure

[0032] 2.2 Purification of the first round of PCR products Remove the AMPure XP Beads (seller: Beckman; product number: A63881, hereinafter the same) 30 minutes in advance to allow them to reach room temperature.

[0033] (1) Mix the same sample prepared in step 2.1 with Nipah A Panel Mix and Nipah B Panel Mix to obtain PCR reaction solutions in a volume ratio of A:B=1:1 (i.e., take 30 μL from tube A and 30 µL from tube B) to form a PCR tube of 60 μL; add 1.5 times the volume of AMPure XP Beads (90 μL) to the mixture, mix thoroughly, and let stand at room temperature for 5 min.

[0034] (2) Place the PCR tube on a magnetic rack and let it stand for 2-5 minutes until the solution is completely clear and transparent. Use a pipette to carefully aspirate the supernatant and discard it.

[0035] (3) Keep the PCR tube on the magnetic rack, add 160 µL of freshly prepared 80% ethanol to rinse the magnetic beads, let stand for 30 seconds, then carefully aspirate and discard the supernatant. Repeat the washing step once, and use a small-capacity pipette to aspirate the liquid at the bottom of the tube.

[0036] (4) Dry at room temperature until the surface of the magnetic beads is no longer reflective and no cracks.

[0037] (5) Remove the PCR tube from the magnetic rack, add 50 µL of TE buffer, vortex to mix, and incubate at room temperature for 5 minutes.

[0038] (6) Place the PCR tube on a magnetic rack and let it stand for 2-5 minutes. After the liquid has cleared, carefully transfer 45 µL of the elution supernatant to a brand new PCR tube, which is the purified first-round PCR product.

[0039] (7) Take 1-2 µL of the product and determine its concentration using the Qubit dsDNA HS high-sensitivity real-time fluorescence kit (vendor: Invitrogen, catalog number: Q32854). The sample concentration should be ≥5 ng / µL.

[0040] 3. Highly efficient enzymatic disruption and end repair 3.1 System configuration and reaction conditions The reaction system is shown in Table 5. The end-break repair reaction solution was prepared on ice using the MGIEasy Fast PCR-FREE enzyme digestion library preparation module (catalog number: 940-000020-00).

[0041] Take a new PCR tube and pipette 15 µL of the end-break repair reaction solution into the tube; according to step 2.2 (7), take 200 ng of PCR product into the tube. Add TE buffer to a volume of 60 µL, vortex 3 times, and centrifuge briefly to collect the reaction solution to the bottom of the tube and place it on ice.

[0042] Set the reaction conditions according to Table 6. Once the temperature of the first step drops to 4°C, place the sample in the PCR instrument and skip the first step to proceed with the reaction.

[0043] Table 5 Preparation system of end-of-phase repair reaction solution

[0044] Table 6 Conditions for Disrupting the End-of-Line Repair Reaction

[0045] 3.2 Disruption of product purification Remove the AMPure XP Beads 30 minutes in advance to allow them to reach room temperature.

[0046] (1) Add 60 µL of AMPure XP Beads to the PCR tube end repair product, mix thoroughly, and let stand at room temperature for 5 min.

[0047] (2) Place the PCR tube on a magnetic rack and let it stand for 2-5 minutes until the solution is completely clear and transparent. Use a pipette to carefully aspirate the supernatant and discard it.

[0048] (3) Keep the PCR tube on the magnetic rack, add 160 µL of freshly prepared 80% ethanol to rinse the magnetic beads, let stand for 30 seconds, then carefully aspirate and discard the supernatant. Repeat the washing step once, and use a small-capacity pipette to aspirate the liquid at the bottom of the tube.

[0049] (4) Dry at room temperature until the surface of the magnetic beads is no longer reflective and no cracks.

[0050] (5) Remove the PCR tube from the magnetic rack and add 45 µL of TE buffer to elute the DNA. The product does not need to be magnetically adsorbed or the supernatant transferred; proceed directly to the next reaction with the magnetic beads.

[0051] 4. Connector connection 4.1 System configuration and reaction conditions Refer to the reagent kit vendor: MGIEAsy Fast PCR-FREE Enzyme Digestion Library Preparation Module (Catalog No.: 940-000020-00). Take out the reaction solution and adapters in advance, and prepare the adapter ligation reaction solution according to Table 7. Prepare the reaction solution on ice, vortex, briefly centrifuge, and then place on ice.

[0052] Add 30 µL of the prepared adapter ligation reaction solution to 45 µL of the end-repair suspension with magnetic beads prepared in step 3.2, and then add 5 µL of adapter (select the appropriate adapter according to the type of sequencer used). In this test, the MGIEAsy paired-end independent tag PF adapter kit (catalog number: 940-000023-00) manufactured by BGI Genomics was used, with a total volume of 80 µL. After vortexing and mixing, briefly centrifuge and proceed with the reaction according to the reaction conditions in Table 8.

[0053] Table 7. Preparation system of reaction solution for connector connection

[0054] Table 8. Connector Connection Reaction Conditions

[0055] 4.2 Purification of Ligation Products Remove the AMPure XP Beads 30 minutes in advance to allow them to reach room temperature.

[0056] (1) Add 27 µL of AMPure XP Beads to the PCR tube ligation product, mix thoroughly, and let stand at room temperature for 5 min.

[0057] (2) Place the PCR tube on a magnetic rack and let it stand for 2-5 minutes until the solution is completely clear and transparent. Use a pipette to carefully aspirate the supernatant and discard it.

[0058] (3) Keep the PCR tube on the magnetic rack, add 160 µL of freshly prepared 80% ethanol to rinse the magnetic beads, let stand for 30 seconds, then carefully aspirate and discard the supernatant. Repeat the washing step once, and use a small-capacity pipette to aspirate the liquid at the bottom of the tube.

[0059] (4) Dry at room temperature until the surface of the magnetic beads is no longer reflective and no cracks.

[0060] (5) Remove the PCR tube from the magnetic rack, add 20 µL of TE buffer to wash out DNA, vortex to mix, and incubate at room temperature for 5 minutes.

[0061] (6) Place the PCR tube on a magnetic rack and let it stand for 2-5 minutes. After the liquid has cleared, carefully transfer 18 µL of the elution supernatant into a brand new PCR tube.

[0062] (7) Take 1 µL of the product and determine its concentration using the Qubit dsDNA HS fluorescence quantitative kit. The sample concentration should be ≥0.8 ng / µL.

[0063] 5. Library preparation and computer-aided testing Prepare the corresponding libraries according to the requirements of the sequencer used, such as the transformation vector system corresponding to the DNBSEQ platform or Illumina platform sequence from BGI Genomics, and then perform sequencing.

[0064] 6. Sequencing and data analysis 6.1 Sequencing Requirements Data volume for computer use: 1 - 5 million reads.

[0065] Sequencing read length: at least 100bp per end.

[0066] The required output for data analysis is ≥0.1 Gb / Sample. The whole genome is assembled using a reference genome.

[0067] 6.2 Bioinformatics Analysis The Viral ConsensusGenomes module was run on the open-source cloud analytics platform CZID (https: / / czid.org / ). The raw fastaq file was uploaded, and the Minimap2 pipeline was used to align the data with the reference genome. Trim galore was used to filter and quality control the uploaded data. Minimap2 then performed further alignment on 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.

[0068] 7. To verify the feasibility and superiority of the kit of this invention, the sequencing coverage and depth of reference strains and clinical samples of different genotypes (B type and M type) were compared and analyzed. The results are as follows: (1) Coverage, depth and sensitivity Using the multiplex amplification system designed in this invention, eight samples each of the Nipah virus reference strains (type B: GenBank-AY988601.X; type M: NC_002728.X) with broad Ct values ​​(15-37), novel coronavirus nucleic acid, and an enzyme-free water blank control were used as templates for library construction and sequencing. The results are shown in Table 10. For samples with Ct values ​​between 15 and 30, regardless of whether they are type B or type M, this kit can achieve 100% Nipah virus whole-genome sequencing coverage, with sequencing depths typically reaching tens of thousands of times (ranging from 15,000x to 49,000x). Particularly noteworthy is that for extremely low-titer samples with Ct values ​​> 35 (such as Ct 36.81 and Ct 37.82), the kit still exhibits extremely sensitive targeted capture efficiency. Except for a very few samples where the coverage dropped to 91.2%, the genome coverage of most ultra-low titer samples remained stable between 97.3% and 100%.

[0069] (2) Application Examples - Clinical Empirical Comparison of Low Viral Load Samples To comprehensively verify the practical performance of the "Nipah virus whole genome targeted amplification and sequencing kit compatible with multiple variants" described in this invention in the front line of public health, especially its genome recovery capability in dealing with low-titer, high-difficulty pathological samples, two real respiratory swab extracts that were medically confirmed to be infected and showed low-titer, weakly positive results in quantitative real-time PCR were selected: ●Sample A: Infected with Nipah virus Bangladeshi strain (NiV-B), Ct value 31.5.

[0070] ●Sample B: Infected with Nipah virus Malaysian strain (NiV-M), Ct value 28.2.

[0071] The experimental control group used a conventional, industry-standard metagenomics library preparation and sequencing (mNGS) process for clinical pathogens without any targeted enrichment, such as the IDseq™ metagenomics sequencing kit from MicroGene. The library sequences generated by both library preparation methods were ultimately used on the same sequencing platform (such as the DNBSEQ series) to generate an equal amount of sequencing data (20,000 valid reads were standardized and aligned). The actual test comparison data is shown in Table 11.

[0072] The detailed data in Table 11 clearly demonstrates that conventional metagenomic methods, when faced with clinical specimens with high Ct values ​​(low gene load), only yielded 18.4% fragmented gene fragments in sample A. The kit and library construction method specified in this invention, through specific physical amplification isolation using a shingled A / B panel pool, achieved 98.6% genome coverage for NiV-B type and 99.5% genome coverage for NiV-M type samples under extreme stress testing with only a small number of sequencing reads. This experimental data effectively proves the superior sensitivity and coverage of the kit of this invention.

[0073] Table 9 Primer Pool Sequences ; ; ; ; ; ;

[0074] Table 10. Broad Ct value sequencing validation results for Nipah virus samples

[0075] Table 11 Comparison of sequencing performance parameters between metagenomic sequencing (mNGS) and tNGS of low-cytotoxicity Nipah virus samples .

Claims

1. Primers for whole-genome sequencing of Nipah virus, characterized in that, The sequences of the primers are shown in SEQ ID NO.1 to SEQ ID NO.

189.

2. A Nipah virus whole genome sequencing kit, characterized in that, It contains the primers as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The primers are divided into Nipah A Panel Mix and Nipah B Panel Mix. The sequence of Nipah A Panel Mix is ​​shown in SEQ ID NO.1 to SEQ ID NO.96, and the sequence of Nipah B Panel Mix is ​​shown in SEQ ID NO.1, SEQ ID NO.97 to SEQ ID NO.

189.

4. The reagent kit according to claim 2, characterized in that, The kit also includes a reverse transcription system, an amplification system, and a product purification system.

5. The application of the primers of claim 1 or the kits of any one of claims 2-4 in Nipah virus whole genome sequencing.

6. A method for whole-genome sequencing of Nipah virus, characterized in that, The process includes the following steps: PCR amplification of the sample to be sequenced using the primers described in claim 1, followed by enzymatic fragmentation and end repair, adapter ligation, library preparation, and sequencing.

7. The method according to claim 6, characterized in that, The PCR amplification system is as follows: Tube A: 4 μL Nipah A Panel Mix, 10 μL first-strand cDNA template, 10 μL 3×T Enzyme, and 6 μL Nuclease-free water; Tube B: 4 μL Nipah B Panel Mix, 10 μL first-strand cDNA template, 10 μL 3×T Enzyme, and 6 μL Nuclease-free water; The sequence of the Nipah A Panel Mix is ​​shown in SEQ ID NO.1~SEQ ID NO.96, and the sequence of the Nipah B Panel Mix is ​​shown in SEQ ID NO.1, SEQ ID NO.97~SEQ ID NO.

189.

8. The method according to claim 7, characterized in that, The PCR product was purified after the A tube and the B tube were mixed.

9. The method according to claim 6, characterized in that, The PCR amplification program is as follows: 98℃ for 3 min; 98℃ for 30 sec, 60℃ for 2 min, 72℃ for 1 min, 25 cycles; 72℃ for 1 min; 4℃ Hold.

10. The method according to claim 6, characterized in that, The sequencing requirements are: 1-5 million reads, sequencing read length: at least 100 bp at one end, and data analysis output ≥ 0.1 Gb / Sample.

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