A method and kit for detecting nipah virus whole genome

Abstract

The application provides a method and a kit for detecting a full genome of a Nipah virus. Specifically, the method comprises detecting the Nipah virus in a sample to be tested by using a PCR amplification reaction system comprising a primer set specific to the Nipah virus (covering the full genome of the Nipah virus) and double-response magnetic beads (the surface of the magnetic beads is modified with a probe specific to the Nipah virus, a temperature-sensitive polymer, and a pH-sensitive group), and synchronously performing PCR amplification and magnetic bead capture, thereby significantly improving the specificity, sensitivity and detection efficiency of virus detection.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, and more specifically, this invention relates to a method and kit for detecting the whole genome of Nipah virus. Background Technology

[0002] Nipah virus (NiV) belongs to the genus Henipavirus in the family Paramyxoviridae. It is a highly pathogenic zoonotic virus.

[0003] Nipah virus can cause severe damage to the central nervous system and respiratory symptoms.

[0004] Currently, genome analysis of highly pathogenic pathogens primarily employs metagenomic sequencing or single PCR amplification sequencing techniques. However, metagenomic sequencing is susceptible to interference from host nucleic acid background in samples with low viral loads, resulting in low target sequence abundance and low effective data utilization. Traditional single PCR or simple multiplex PCR often suffers from uneven amplification coverage, missing genome terminal sequences, and primer interference, making it difficult to achieve high-completeness assembly of the entire genome. Especially for high CT value, low copy number samples, conventional methods often fail to obtain complete genome sequences, severely restricting the development of Nipah virus mutation monitoring, molecular tracing, and emergency detection technology reserves. Furthermore, magnetic nanoparticle enrichment technology, due to its advantages of high specificity, ease of operation, and ease of automated integration, has been widely used in nucleic acid purification and target enrichment. However, existing magnetic nanoparticles are mostly general-purpose, exhibiting problems such as low binding efficiency, strong non-specific adsorption, and insufficient elution efficiency.

[0005] Therefore, developing a safe and efficient whole-genome detection technology for Nipah virus to address the technical challenges of target sequence enrichment and low assembly integrity in low viral load samples is of great practical significance and application value for improving my country's ability to respond to emerging and re-emerging infectious diseases. It also provides new ideas and methods for the development of molecular detection technologies for highly pathogenic viruses. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting the whole genome of Nipah virus with low viral load.

[0007] In a first aspect of the present invention, a method for detecting Nipah virus is provided, the method comprising the steps of: (1) Provide a sample to be tested, obtain the RNA of the sample and reverse transcribe it to obtain cDNA; (2) The cDNA is amplified in a PCR amplification reaction system containing a Nipah virus-specific primer set and dual-response magnetic beads, and the cDNA amplification product is captured by the magnetic beads. The primer set includes: Primer set 1: forward primers as shown in SEQ ID NO. 1-32; and reverse primers as shown in SEQ ID NO. 33-62; and Primer set 2: forward primers as shown in SEQ ID NO. 63-89; and reverse primers as shown in SEQ ID NO. 90-115; The surface of the dual-response magnetic beads is modified with a Nipah virus-specific probe, a thermosensitive polymer, and a pH-sensitive group, as shown in SEQ ID NO.116. (3) Detect the cDNA amplification product.

[0008] In another preferred embodiment, primer set 1 and primer set 2 are placed in two tubes of PCR amplification reaction system, and the reaction system is identical in all components except for the primer sets.

[0009] In another preferred embodiment, the magnetic beads are selected from Fe3O4 magnetic beads, cobalt ferrite, Fe3O4@SiO2 core-shell magnetic beads, etc.

[0010] In another preferred embodiment, the magnetic beads have a particle size of 100-200 nm, preferably 130-180 nm, and more preferably 150 nm.

[0011] In another preferred embodiment, the temperature-sensitive polymer is selected from PNIPAM monomers, N-vinylcaprolactam, N-tert-butylacrylamide, etc.

[0012] In another preferred embodiment, the pH-sensitive group is selected from aminoacetic acid, carboxyl, imidazole, pyridine, dimethylamino, etc.

[0013] In another preferred embodiment, the sample to be tested is selected from: respiratory samples (nasopharyngeal swabs, sputum, bronchoalveolar lavage fluid (BALF), throat swabs, nasal wash, tracheal aspirate), serum samples (whole blood, serum, plasma), and cerebrospinal fluid samples.

[0014] In another preferred embodiment, the Nipah virus is selected from the Bengal type (NIV-B) and the Malaysian type (NIV-M).

[0015] In another preferred embodiment, the PCR amplification and magnetic bead capture occur simultaneously.

[0016] In another preferred embodiment, step (2) further includes: after the magnetic beads capture the cDNA amplification product, washing the non-specific adsorbed and background nucleic acids with a buffer solution.

[0017] In another preferred embodiment, step (2) further includes: releasing the cDNA amplification product coupled to the magnetic beads under acidic conditions.

[0018] In another preferred embodiment, the acidic conditions are pH 5.0-5.5.

[0019] In another preferred embodiment, the magnetic bead can be reused at least three times.

[0020] In another preferred embodiment, step (3) includes performing gel electrophoresis on the cDNA amplification product and comparing it with the target gene to detect the presence of the Nipah virus genome.

[0021] In a second aspect of the invention, a Nipah virus-specific primer set is provided, the primer set comprising: Primer set 1: forward primers as shown in SEQ ID NO. 1-32; and reverse primers as shown in SEQ ID NO. 33-62; and Primer set 2: forward primers as shown in SEQ ID NO.63-89; and reverse primers as shown in SEQ ID NO.90-115.

[0022] In another preferred embodiment, the primer set covers the entire genome of Nipah virus.

[0023] In a third aspect of the invention, a Nipah virus-specific magnetic bead is provided, the surface of which is modified with a Nipah virus-specific probe, a temperature-sensitive polymer, and a pH-sensitive group as shown in SEQ ID NO. 116.

[0024] In another preferred embodiment, the magnetic beads are selected from Fe3O4 magnetic beads, cobalt ferrite, Fe3O4@SiO2 core-shell magnetic beads, etc.

[0025] In another preferred embodiment, the magnetic beads have a particle size of 100-200 nm, preferably 130-180 nm, and more preferably 150 nm.

[0026] In another preferred embodiment, the temperature-sensitive polymer is selected from PNIPAM monomers, N-vinylcaprolactam, N-tert-butylacrylamide, etc.

[0027] In another preferred embodiment, the pH-sensitive group is selected from aminoacetic acid, carboxyl, imidazole, pyridine, dimethylamino, etc.

[0028] In a fourth aspect of the invention, a kit is provided, the kit comprising: 1) The Nipah virus-specific primer set according to the second aspect of the present invention, the primer set comprising: Primer set 1: forward primers as shown in SEQ ID NO. 1-32; and reverse primers as shown in SEQ ID NO. 33-62; and Primer set 2: forward primers as shown in SEQ ID NO. 63-89; and reverse primers as shown in SEQ ID NO. 90-115; 2) The Nipah virus-specific magnetic beads of the third aspect of the present invention, wherein the surface of the magnetic beads is modified with a Nipah virus-specific probe, a temperature-sensitive polymer, and a pH-sensitive group as shown in SEQ ID NO. 116.

[0029] In another preferred embodiment, the kit further includes other components for PCR amplification, such as PCR mix and buffer.

[0030] In another preferred embodiment, the kit further includes an RNA reverse transcription reagent.

[0031] In another preferred embodiment, the kit further includes a lysis buffer and an elution buffer.

[0032] In another preferred embodiment, the kit also includes instructions for guiding Nipah virus gene detection.

[0033] In a fifth aspect of the invention, the use of the primer set described in the second aspect of the invention, the magnetic beads described in the third aspect of the invention, and / or the kit described in the fourth aspect of the invention is provided for the detection of Nipah virus.

[0034] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0035] Figure 1 Agarose gel electrophoresis verification of the enriched products of the entire Nipah virus genome was shown.

[0036] Figure 2 The results show the validation of genome coverage for Nipah virus samples with different viral loads. Detailed Implementation

[0037] Through extensive and in-depth research, the inventors have developed a method for detecting the whole genome of Nipah virus based on multiplex PCR coupled with magnetic nanobead enrichment. This invention utilizes 47 sets of wavy PCR multiplex primers, ensuring 20-50 bp overlap between adjacent primer amplification products, achieving comprehensive amplification of the entire genome without blind spots. Sequence comparison and screening eliminate non-specific interference from background nucleic acids. Furthermore, by developing magnetic nanobeads surface-modified with temperature-sensitive polymer (PNIPAM), pH-sensitive groups, and Nipah virus-specific capture probes, multiplex PCR pre-amplification is coupled with specific enrichment by the magnetic nanobeads, constructing an integrated "simultaneous amplification-simultaneous capture-response elution" process, improving capture efficiency and reducing costs. Results show that the proposed detection method can amplify and capture high-quality Nipah virus whole genome sequences, significantly improving the sensitivity and accuracy of gene detection. This invention was completed based on these findings.

[0038] the term To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0039] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0040] Nipah virus Nipah virus (NiV) is a highly lethal zoonotic RNA virus belonging to the genus Henipavirus in the family Paramyxoviridae. It is a single-stranded, negative-sense RNA virus with a genome length of approximately 18.2 kb. The virus particle is pleomorphic, with a diameter of 150-300 nm. The viral genome encodes six structural proteins and three non-structural proteins. The outermost layer is a lipoprotein envelope, and it is sensitive to heat and common disinfectants. Based on differences in genomic sequence, it is mainly divided into two genotypes: the Malaysian strain (NiV-M) and the Bangladeshi strain (NiV-B).

[0041] Natural host: Fruit bats of the family Pteropodidae are the natural host of the virus. Bats infected with the virus usually do not develop symptoms.

[0042] Transmission routes: Animal-to-human transmission: direct contact with infected animals (such as pigs or horses) or their bodily fluids.

[0043] Food contamination: Consuming fruits or other foods contaminated with the saliva or urine of infected bats.

[0044] Human-to-human transmission: spread among family members or healthcare workers through close contact with the patient’s bodily fluids (such as blood, urine, nasopharyngeal secretions).

[0045] Incubation period: usually 3 to 14 days, but in rare cases it can be up to 45 days.

[0046] Symptoms: Infection may be asymptomatic, but most patients will experience fever, headache, cough, sore throat, and difficulty breathing. In severe cases, it can rapidly develop into encephalitis (brain swelling), leading to confusion, drowsiness, seizures, and coma within 24-48 hours. About one-fifth of survivors will suffer from long-term neurological sequelae.

[0047] Diagnosis mainly relies on PCR detection of viral RNA in respiratory samples, blood, or cerebrospinal fluid.

[0048] Nipah virus detection methods Based on the conservation of the whole genome sequence and the distribution of structural regions of Nipah virus, this invention screened conserved regions from 424 Nipah virus reference sequences downloaded from the NCBI gene database (https: / / www.ncbi.nlm.nih.gov / ), designed and optimized 47 sets of specific multiplex PCR primers, and established a highly specific and highly uniform multiplex PCR amplification system by systematically optimizing primer concentration, reaction buffer system and primer ratio. This system can simultaneously amplify multiple target fragments of the whole Nipah virus genome in a single reaction tube, effectively avoiding primer dimers, non-specific amplification and regional coverage deviation.

[0049] Meanwhile, this invention develops temperature / pH dual-responsive functionalized magnetic nanobeads for Nipah virus. A Nipah virus-specific capture probe (SEQ ID NO.116), a temperature-sensitive polymer (PNIPAM), and a pH-sensitive group (aminoacetic acid) are coupled to the surface of the magnetic beads. The particle size and surface modification process of the magnetic beads are optimized to improve the binding efficiency of the magnetic beads with Nipah virus amplification products and the stability of the magnetic beads themselves.

[0050] This novel magnetic nanobead is deeply coupled with the multiplex PCR amplification process to achieve "synchronous PCR amplification and magnetic bead capture, and responsive regulation of the elution process". By utilizing the specific interaction between the magnetic bead and the target sequence and the magnetic field for rapid separation, it achieves efficient enrichment and purification of viral nucleic acid fragments, and significantly removes host nucleic acid, enzyme inhibitors, primer residues and impurities.

[0051] Thermosensitive polymer The thermosensitive polymer used in this invention is poly(N-isopropylacrylamide) (PNIPAM), whose carbon backbone side chains possess both hydrophilic amide groups and hydrophobic isopropyl groups. PNIPAM has a typical lowest critical solution temperature (LCST, approximately 32°C): below the critical temperature, the amide bonds form hydrogen bonds with water molecules, and the polymer chains extend into a hydrophilic swelling state; above the critical temperature, the hydrogen bonds dissociate, the hydrophobic group effect is enhanced, and the molecular chains coil and aggregate, exhibiting hydrophobicity. Utilizing this reversible temperature-induced phase transition characteristic, the interfacial properties of the magnetic bead surface can be controlled by temperature variations, enabling controlled capture and elution of analytes. In addition to PNIPAM, the thermosensitive polymer can also be selected from any one or more copolymers of N-vinylcaprolactam (PVCL), N,N-dimethylaminoethyl methacrylate (PDMAEMA), polyethylene glycol methacrylate polymer (POEGMA), N-tert-butylacrylamide polymer, and N-isopropylmethacrylamide.

[0052] pH-sensitive groups This invention utilizes glycine as a pH-sensitive functional group. The glycine molecule contains both amino and carboxyl amphoteric functional groups. In systems with varying pH levels, the amino group can undergo protonation, and the carboxyl group can undergo dissociation, allowing the modified support to carry different electrical charges. Electrostatic repulsion induces the swelling or contraction of the polymer layer, enabling pH-controlled regulation of the magnetic bead interface charge and adsorption performance. This, combined with a temperature-sensitive polymer, constructs a dual-response magnetic separation support. Besides glycine, the pH-sensitive group also includes one or more combinations of carboxyl, sulfonic acid, imidazole, pyridinyl, dimethylamino, maleic acid, and histidine groups.

[0053] Compared with the prior art, the main advantages of the present invention include: 1. Specific Primers: This invention designs 47 sets of genome-wide specific primers, employing a shingled design to ensure that the amplification products of adjacent primers overlap by 20-50 bp, achieving comprehensive coverage of the entire genome. Simultaneously, through sequence alignment and screening, it is ensured that the primers target and bind only to the target viral RNA, effectively avoiding non-specific interference from host nucleic acid background and other viruses, thus fundamentally solving the technical problem of insufficient viral genome enrichment specificity in existing technologies.

[0054] 2. Dual-responsive magnetic nanobeads: The temperature / pH dual-responsive functionalized magnetic nanobeads developed in this invention use Fe3O4 as the core, with the surface modified with a temperature-sensitive polymer (PNIPAM) and pH-sensitive groups, and are coupled with a Nipah virus-specific capture probe. The magnetic bead particle size (100-200nm, average 150nm) and surface modification process are optimized, which improves the binding efficiency of the magnetic beads by more than 30% and the stability by 40% compared with conventional general-purpose magnetic beads. The specific binding rate is ≥98%, and the non-specific binding rate is ≤2%. It can achieve efficient enrichment in low-copy simulated samples with CT values ​​of less than 34, significantly improving the target concentration and sequencing data utilization.

[0055] 3. Coupling Mechanism: This invention couples multiplex PCR pre-amplification with specific enrichment of magnetic nanoparticles, constructing an integrated workflow of "simultaneous amplification-simultaneous capture-response elution," breaking the conventional step-by-step operation mode of "PCR amplification first, then magnetic bead capture," and shortening the operation time (40% shorter than conventional methods). Simultaneously, it enables the recovery and reuse of magnetic beads (capture efficiency remains ≥85% after 3 reuses), reducing experimental costs.

[0056] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0057] Example 1. Confirmation of Nipah virus experimental samples and whole genome information This embodiment uses Nipah virus whole-genome pseudoviruses (NIV-M and NIV-B) as experimental samples to simulate the detection and enrichment of Nipah virus nucleic acid in clinical samples. Sequencing verified that the target sequence was correct and there was no bacterial contamination. Specific sample information and basic test records are as follows.

[0058] 1.1 Basic Information of the Sample A total of 12 pseudovirus samples were used in the experiment, covering two genotypes (NIV-M pseudovirus and NIV-B pseudovirus). For each genotype, a high-concentration group (Ct value ≈ 25, simulating clinical high-viral-load samples) and a low-concentration group (Ct value ≈ 34, simulating clinical low-viral-load samples) were set up. Each group had three biological replicates to ensure the reliability of the experimental results. Specific information on the samples and Ct value detection records are shown in Table 1. All samples were stored in an ultra-low temperature freezer at -80℃ and thawed slowly on ice before use to avoid repeated freeze-thaw cycles that could lead to nucleic acid degradation.

[0059] Table 1. Information on Nipah virus pseudovirus samples and Ct value detection records Note: All sample coefficients of variation (CV) are <1%, indicating good sample homogeneity, which can be used for subsequent experiments.

[0060] 1.2 Nucleic acid extraction from samples Total RNA (Nipah virus whole genome nucleic acid samples) from the above 12 pseudovirus samples was extracted using a magnetic bead nucleic acid extraction kit (purchased from Tiangen Biotech Co., Ltd., catalog number: DP301) in accordance with the kit instructions.

[0061] Extraction quality verification: The purity and concentration of extracted nucleic acids were detected using a Nanodrop 2000 nucleic acid analyzer. The verification standards were: an A260 / A280 ratio of 1.8–2.0 (acute nucleic acid purity), and a nucleic acid concentration ≥50 ng / μL (meeting the requirements for subsequent reverse transcription and PCR amplification). The test results showed that the A260 / A280 ratios of the 12 samples ranged from 1.85 to 1.92, and the concentrations ranged from 52 to 68 ng / μL, all meeting the verification standards. There was no degradation or protein contamination, and the samples were suitable for subsequent experiments.

[0062] Example 2. Primer design for Nipah virus enriched swathenoPCR targeting whole-genome nucleic acid targets. All publicly available Nipah Virus (NiV) whole genome sequences, totaling 424, were downloaded from the NCBI Genome Database (https: / / www.ncbi.nlm.nih.gov / ). These sequences cover major circulating strains such as the Bengal type (NIV-B) and the Malaysian type (NIV-M), as well as variants isolated from different regions. Software was used to remove sequence duplication (sequences with similarity ≥99% were considered repetitive), and clustering was established based on sequence similarity. Multiple sequence alignment was performed, and primers for the whole genome were designed using a shingled primer pattern, ensuring that the amplification products of adjacent primer pairs overlapped within 20-50 bp, thus achieving full coverage of the Nipah Virus genome sequence. Multiple factors were considered when selecting primer pairs, including PCR product length, melting temperature, and dimerization.

[0063] This application designs a Nipah virus-specific primer set, comprising primer set 1 (Panel 1) and primer set 2 (Panel 2), with a target product of approximately 450-600 bp. The sequence information of primer set 1 (Panel 1) is shown in Table 2, and the sequence information of primer set 2 (Panel 2) is shown in Table 3. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a purity ≥99%, and were lyophilized. Before use, they were dissolved in 1×TE buffer to prepare a 10 μmol / L stock solution, which was then stored at -20℃ for later use.

[0064] Table 2 Nipah virus primer set 1 Table 3 Nipah virus primer set 2 In the primer sequences above, A represents adenine, G represents guanine, C represents cytosine, T represents thymine, the degenerate base R represents A / G, Y represents C / T, M represents A / C, W represents A / T, K represents G / T, and D represents A / G / T.

[0065] Primer design instructions: In primer sequences, multiple primer pairs are designed for a specific region, which differs from the conventional single-primer amplification mode. This approach possesses multiple unique technical features, precisely addressing the challenge of amplifying highly variable segments of the Nipah virus genome. Taking the NIV_30 primer as an example, the specific design features are as follows: 1) Multi-layered fault-tolerant coverage of high mutation hotspots. Taking the NIV_30 primer as an example, it corresponds to a high-frequency mutation region in the Nipah virus genome. This region exhibits significant base differences among mainstream Malaysian and Bangladeshi strains and regional variants, with frequent single or multiple mutations. Using a single primer easily leads to primer annealing failure and missing amplification of the target fragment. This invention simultaneously designs three upstream primers and two downstream primers at this site, constructing a multi-binding-site complementary amplification system. By using multiple primers to cover various mutation profiles in this region layer by layer, it avoids amplification blind spots caused by strain mutations with a single primer, achieving full coverage amplification of the high-variability region.

[0066] 2) Multiple overlapping amplified fragments ensure the integrity of the whole genome assembly. Multiple primer pairs for the NIV_30 site generate overlapping amplified products. Compared to single primer pairs producing only a single amplified fragment, multi-layered nested amplification allows for multiple fragment overlaps in this highly variable region. During subsequent whole-genome sequencing and assembly, these overlapping fragments can be used to correct sequence information and fill amplification gaps, preventing genome splicing gaps and sequence distortion caused by the loss of a single fragment in highly variable regions. This ensures the complete and accurate assembly of the Nipah virus full-length genome.

[0067] 3) Improved sensitivity for amplification of low viral load samples. Addressing the challenges of low template abundance and weak amplification signals in clinically latent low viral load samples (Ct value ≈ 34), the NIV_30 multi-set nested primers enable simultaneous amplification of multiple targets within the same region in a multiplex PCR system. This increases the binding probability of template molecules to primers, strengthens the amplification output of this weak region, and allows for stable and complete amplification products from both high and low viral load samples, thus improving the overall sensitivity and repeatability of this enrichment detection system.

[0068] Example 3. Synthesis of Nipah virus cDNA (1) Prepare the cDNA synthesis mix shown in Table 4 by mixing the extracted Nipah whole genome pseudovirus nucleic acid in a 0.2 ml PCR tube: Table 4 Nipah virus RNA reverse transcription system (2) Gently tap the tube wall to mix, centrifuge briefly, and perform reverse transcription according to the reverse transcription procedure in Table 5: Table 5 Reverse Transcription Procedure Example 4. Preparation of Dual-Response Functionalized Magnetic Nanobeads Preparation of dual-responsive functionalized magnetic nanobeads targeting Nipah virus This invention does not use conventional, general-purpose magnetic nanobeads. Instead, it designs and prepares temperature / pH dual-responsive functionalized magnetic nanobeads specifically targeting the sequence characteristics of the Nipah virus genome. These magnetic beads are the core for achieving "precise coupling and efficient enrichment." The specific preparation method and performance characteristics are as follows. The experimental procedures were conducted in a sterile laminar flow hood: 1. Preparation of magnetic bead carriers Using Fe3O4 as the core, a solvothermal method was employed to prepare Fe3O4 nanobeads. 0.8 g of FeCl3·6H2O and 2.0 g of sodium acetate were dissolved in 40 mL of ethylene glycol and reacted at 180 °C for 12 h. After centrifugation, the nanobeads were washed alternately with anhydrous ethanol and deionized water, and then vacuum-dried at 60 °C for 8 h to obtain the Fe3O4 nanobead core. TEM analysis showed that the particle size of the magnetic beads was controlled to be 100-200 nm (average 150 nm), with a CV ≤ 15%. VSM analysis showed that their saturation magnetization was ≥ 80 emu / g, exhibiting both good magnetic responsiveness and low nonspecific adsorption.

[0069] 2. Dual-response layer modification 0.1 g of Fe3O4 magnetic bead cores were ultrasonically dispersed in 30 mL of deionized water. 0.5 g of PNIPAM monomer and 0.02 g of crosslinking agent were added, followed by nitrogen purging and the addition of 0.01 g of initiator. Polymerization was carried out at 60 °C for 4 h. After washing and drying, temperature-sensitive modified magnetic beads were obtained. These beads were dispersed in 25 mL of 0.1 mol / L MES buffer (pH 6.0), activated by EDC / NHS, and then 0.1 g of glycine was added. The reaction was carried out at room temperature for 6 h. After washing and drying, dual-responsive magnetic beads were obtained. The LCST of PNIPAM was controlled at 55-60 °C (average 58 °C), matching the PCR extension and elution temperatures. The pKa of the pH-sensitive group was controlled at 5.0-5.5 (average 5.2), enabling rapid dissociation of the target sequence under acidic conditions and avoiding nucleic acid degradation.

[0070] 3. Nipah virus-specific probe modification This invention involves coupling a Nipah virus genome conserved region-specific probe to the surface of a dual-response capsid. The Nipah virus N gene, located at the uppermost part of the genome, is the core target with the highest conservation and lowest mutation rate. The invention includes 47 sets of Nipah virus whole-genome PCR primers, which can amplify the viral genome into 47 specific fragments of 450bp-600bp, with one set of primers corresponding to the N gene segment. This invention designs a specific probe targeting the 100-250bp site of the conserved region of the Nipah virus N gene, with the sequence 5'-NH2-TTGCTGCTGCTGCTGCTGCT-3' (SEQ ID NO. 116), 20bp in length. This probe only pairs complementaryly with the internal sequence of the 450-600bp amplification fragment corresponding to the N gene in this invention, and has no overlap with any of the 47 primer sets, effectively avoiding competitive binding between primers and probes and not affecting normal whole-genome amplification.

[0071] Specific modification steps: Take 0.1g of dual-responsive magnetic beads, disperse them in 20mL of 0.1mol / L PBS buffer (pH 7.4), sonicate for 10min, add 0.02g EDC and 0.01g NHS, and activate the carboxyl groups on the surface of the magnetic beads for 30min; add 10μL of a 100μmol / L specific probe solution, stir and react at room temperature in the dark for 8h, collect the magnetic beads by centrifugation, wash three times with PBS buffer to remove unbound free probe; detect by ultraviolet spectrophotometry to determine that the probe modification amount is controlled at 50-100pmol / mg magnetic beads (average 75pmol / mg magnetic beads). Specificity verification shows that the magnetic beads specifically bind only to Nipah virus amplification products, and the binding rates with host nucleic acid (human genomic DNA) and primer dimers are both ≤2%. (Among the 47 amplicones, one pair of primers, the first set, specifically amplifies the 100-250 bp region of the N gene. The probe is designed to target this single amplicon. The probe is only complementary to the sequence within this amplification product and does not overlap with any other primer sequence. During PCR, the primers extend first to synthesize the amplicon, and then the probe binds to the amplicon, avoiding competition for primer binding sites and preventing competitive inhibition. The probe does not bind to all 47 amplicons; it only targets a specific amplicon that is part of the set of amplification products from the 47 primer pairs.) 4. The performance comparison between dual-response magnetic beads and conventional magnetic beads is shown in Table 8, indicating that the dual-response magnetic beads of the present invention have significantly better specific binding to the target gene than general magnetic beads.

[0072] Table 8. Performance Comparison of Dual-Response Magnetic Beads and Conventional Magnetic Beads Example 5. Targeted enrichment of the entire Nipah virus genome. This invention employs a one-step gene enrichment strategy that combines simultaneous PCR amplification and magnetic bead capture with responsive regulation of the elution process. This breaks away from the conventional step-by-step operation mode of "PCR amplification first, then magnetic bead capture," shortening the operation time and improving enrichment efficiency. The specific coupling process is as follows (all operations are performed under aseptic conditions, and all reagents are preheated to 25°C): i. Directly add 10 μL of the prepared dual-response functionalized magnetic nanobeads to the multiplex PCR reaction system, vortex for 10 s to mix thoroughly, so that the magnetic beads are evenly dispersed in the reaction system. The final concentration of the magnetic beads is 0.8 mg / mL. No additional capture buffer needs to be prepared or added, which simplifies the operation process and completely avoids the interference of exogenous buffer components on PCR amplification and magnetic bead-specific capture, so as to achieve simultaneous coupling of amplification and capture.

[0073] ii. After the reaction, there is no need for the usual multiple washing steps. Simply add 5 μL of 1 mol / L HCl solution to the coupling system and adjust the pH value of the system to 5.0-5.5. Relying on the dual response characteristics of the magnetic beads, the pH-sensitive groups on the surface undergo protonation and change in charge characteristics. Combined with the temperature-sensitive polymer chain stretching effect, the base complementary binding force between the probe and the viral target sequence is significantly weakened, which promotes the rapid and complete dissociation of the target nucleic acid. The elution efficiency is ≥95%, and there is no obvious degradation of the target sequence. Agarose gel electrophoresis confirms that the bands are clear and without tailing.

[0074] iii. After elution, place the system on a magnetic rack and let it stand for 30 seconds to achieve rapid and complete separation of the magnetic beads from the eluent. The magnetic beads are completely adsorbed onto the tube wall without residue. The supernatant is then aspirated to obtain the high-purity Nipah virus amplification product. Furthermore, the separated magnetic beads can be washed three times with buffer and dried for reuse. After three reuses, the target capture efficiency remains above 85%, effectively reducing experimental costs.

[0075] PCR reaction: (1) PCR multiplex amplification According to Table 6, each sample was prepared separately. P1 and P2 two PCR mixes: Table 6 PCR amplification reaction system Close the tube cap, vortex to mix, briefly incubate on a PCR instrument and perform the capture amplification reaction as shown in Table 7: Table 7 Multiplex PCR Amplification Reaction Procedure A 30 μL Nipah virus multiplex PCR amplification reaction system was thoroughly mixed with 10 μL of novel functionalized magnetic nanobeads developed in this invention (with optimized particle size and surface modification, resulting in a binding efficiency more than 30% higher and stability 40% higher than conventional magnetic beads). The surface of these magnetic nanobeads was modified with Nipah virus-specific capture probes. The mixture was incubated at a suitable temperature to allow the target sequence to hybridize with the magnetic bead probes. Magnetic separation was then performed in a magnetic field, and the supernatant was discarded. Washing buffer was added for repeated washing to remove non-specific adsorbed impurities, unbound primers, and host background nucleic acids. Finally, elution buffer was added to obtain a high-purity Nipah virus whole-genome enrichment product. Qubit assay showed that the purity of the enriched product was above 30 ng / μL, representing an 8-10 fold increase in concentration compared to before enrichment.

[0076] In addition, the effects of coupled enrichment (PCR amplification and magnetic bead capture are performed simultaneously) and conventional stepwise enrichment (PCR amplification is performed first and then magnetic bead enrichment) were compared. The results showed that the elution efficiency of the coupled enrichment technology of the present invention was ≥95%, and the target sequence was not significantly degraded. Agarose gel electrophoresis confirmed that the bands were clear and without tailing.

[0077] Table 9. Comparison of Technical Effect Parameters of Different Nucleic Acid Enrichment Methods Example 6. Quality Inspection of the Capture-Enriched Library Taking an NIV pseudovirus sample as an example, after the reaction, the nucleic acid enrichment product and DL2000 marker were subjected to agarose gel electrophoresis for detection. The results are as follows: Figure 1 As shown, compared with the marker, the bands of each target gene are clear.

[0078] Example 7. Sequencing and Data Analysis The amplified library was purified using one volume of magnetic beads. Samples with a Qubit quantification of 10 ng / µl or higher after purification could be directly used for subsequent library construction experiments. Library construction was performed according to the manufacturer's instructions, starting with 0.5 ng of DNA per sample. The purified PCR products were quantified using the Qubit dsDNA BR Assay kit (Thermo Fisher Scientific). The purified library was sequenced using the Illumina Miseq platform in PE150 mode. The split fastq files after sequencing were uploaded to CLC Genomics Workbench for analysis.

[0079] The sequencing data analysis is shown in Table 10. It can be seen that the sequencing quality Q30 of the samples is above 98%, and the number of reads per sample is <0.5M, making it suitable for all mainstream second- or third-generation sequencing platforms.

[0080] During the data analysis phase, basic quality control, such as base quality and GC content, is performed using software. Low-quality data is filtered using software (sequences with adapter contamination, length <50bp, average quality value <20, or N number >3 are removed). Finally, genome assembly is performed to obtain the complete viral genome sequence.

[0081] Analysis results as follows Figure 2 As shown, with a data volume of 100M, the coverage of samples with NIV viral ct values ​​below 34 at 1X, 10X, and 100X all reached over 99.2%; therefore, it meets the requirement that with a sequencing data volume of 100M, 1×10 4 Nipah virus samples with copies / mL (ct value of approximately 34) or higher can be successfully enriched.

[0082] Table 10 Analysis of Next-Generation Sequencing Data discuss: This invention addresses the shortcomings of existing technologies, such as low loading capacity and poor enrichment, incomplete genome coverage, strong host interference, cumbersome operation, high cost, significant biological risks, and weak reproducibility. Through an integrated design of multiple primers for conserved regions using shingled structures, dual-response functionalized magnetic nanobeads, simultaneous PCR-magnetic bead coupling enrichment, and targeted capture with specific probes, it achieves the following substantial beneficial effects: (1) Breaking through the low copy limit, the enrichment efficiency is greatly improved. Overcoming the problem of amplification failure in high Ct value samples using traditional techniques, it can stably adapt to low-load samples with Ct≤34, with a minimum adaptability of 1×10⁻⁶. 4 With copies / mL, the target nucleic acid enrichment fold reaches 8-10 times, significantly improving the detection and enrichment ability of trace samples.

[0083] (2) Complete genome coverage and excellent assembly quality Using 20–50 bp overlapping wavy primers, we achieved full coverage of mainstream M / B type strains, with a genome assembly integrity of ≥99.97%, no gaps, and no terminal deletions, providing complete sequence support for strain tracing and variation monitoring.

[0084] (3) High specificity and extremely low interference from impurities The dual specific screening of primer conserved targeting and magnetic bead-specific probes results in a non-specific binding rate of ≤2% and host nucleic acid contamination of ≤0.5%, which is far superior to the 5%–8% contamination level of conventional magnetic beads. The effective sequencing rate is ≥98%, and the data utilization rate is significantly improved.

[0085] (4) The process is streamlined and efficient, pollution-free and easy to scale up. Breaking away from the bias of stepwise enrichment technology, we adopt a simultaneous amplification-capture coupling process, which reduces the process time from 150 minutes to 90 minutes, a reduction of 40% in steps; single-tube closed operation reduces aerosol contamination and is suitable for batch detection and automated applications.

[0086] (5) Magnetic beads have superior performance, are recyclable, and save costs. Customized 100-200nm dual-response magnetic beads significantly improve magnetization efficiency, probe loading, and elution efficiency; the magnetic beads can be reused 3 times with a capture efficiency of ≥85%, greatly reducing consumables and experimental costs, and making them more widely applicable.

[0087] (6) The system is stable and repeatable, and the results are accurate and controllable. The multiplex PCR system has been optimized with multiple parameters, achieving a biological repeatability CV of <1%, stable nucleic acid purity within the optimal range, sequencing Q30 >98%, and small intra-batch / inter-batch differences, meeting the requirements for standardized testing and scientific research quantification.

[0088] (7) High biosafety and few application restrictions The method validation is completed by relying on whole-genome pseudoviruses to simulate nucleic acids. It does not require contact with BSL-4 wild-type strains, avoids the risks of handling highly pathogenic pathogens, complies with domestic biosafety control standards, and has a wider range of applicable scenarios.

[0089] (8) The platform has strong compatibility and expands the scope of applications. Gentle elution ensures the integrity of nucleic acid fragments, with a library preparation starting amount as low as 0.5ng, and is compatible with mainstream second- and third-generation sequencing platforms; it can be extended to multiple fields such as port quarantine, emergency prevention and control, and zoonotic disease monitoring.

[0090] (9) The nucleic acid integrity is well preserved, which has high value for further research. The entire process is conducted under mild reaction conditions to avoid nucleic acid breakage and degradation, thus preserving the viral mutation region and functional gene information. This provides a high-quality nucleic acid template for subsequent gene function analysis and vaccine target development.

[0091] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0092] NIV-1-1 whole genome sequence (SEQ ID NO.117):

Claims

1. A method for detecting Nipah virus, characterized in that, The detection method described: (1) Provide a sample to be tested, obtain the RNA of the sample and reverse transcribe it to obtain cDNA; (2) The cDNA is amplified in a PCR amplification reaction system containing a Nipah virus-specific primer set and dual-response magnetic beads, and the cDNA amplification product is captured by the magnetic beads. The primer set includes: Primer set 1: forward primers as shown in SEQ ID NO. 1-32; and reverse primers as shown in SEQ ID NO. 33-62; and Primer set 2: forward primers as shown in SEQ ID NO. 63-89; and reverse primers as shown in SEQ ID NO. 90-115; The surface of the dual-response magnetic beads is modified with a Nipah virus-specific probe, a thermosensitive polymer, and a pH-sensitive group, as shown in SEQ ID NO.

116. (3) Detect the cDNA amplification product.

2. The detection method as described in claim 1, characterized in that, The magnetic beads are selected from Fe3O4 magnetic beads, cobalt ferrite, and Fe3O4@SiO2 core-shell magnetic beads.

3. The detection method as described in claim 1, characterized in that, The magnetic beads have a particle size of 100-200nm, preferably 130-180nm, and more preferably 150nm.

4. The detection method as described in claim 1, characterized in that, The thermosensitive polymer is selected from PNIPAM monomer, N-vinylcaprolactam, N-tert-butylacrylamide, etc.

5. The detection method as described in claim 1, characterized in that, The samples to be tested were selected from: respiratory samples (nasopharyngeal swabs, sputum, bronchoalveolar lavage fluid (BALF), throat swabs, nasal wash, tracheal aspirate), serum samples (whole blood, serum, plasma), and cerebrospinal fluid samples.

6. The detection method as described in claim 1, characterized in that, The Nipah virus was selected from the Bengal type (NIV-B) and the Malaysian type (NIV-M).

7. A Nipah virus-specific primer set, characterized in that, The primer set comprises: Primer set 1: forward primers as shown in SEQ ID NO. 1-32; and reverse primers as shown in SEQ ID NO. 33-62; and Primer set 2: forward primers as shown in SEQ ID NO.63-89; and reverse primers as shown in SEQ ID NO.90-115.

8. A Nipah virus-specific magnetic bead, characterized in that, The surface of the magnetic beads is modified with a Nipah virus-specific probe, a thermosensitive polymer, and a pH-sensitive group, as shown in SEQ ID NO.

116.

9. A reagent kit, characterized in that, The kit includes: 1) The Nipah virus-specific primer set of claim 7, wherein the primer set comprises: Primer set 1: forward primers as shown in SEQ ID NO. 1-32; and reverse primers as shown in SEQ ID NO. 33-62; and Primer set 2: forward primers as shown in SEQ ID NO. 63-89; and reverse primers as shown in SEQ ID NO. 90-115; 2) The Nipah virus-specific magnetic beads of claim 8, wherein the surface of the magnetic beads is modified with a Nipah virus-specific probe, a temperature-sensitive polymer, and a pH-sensitive group as shown in SEQ ID NO.

116.

10. Use of the primer set as claimed in claim 7, the magnetic beads as claimed in claim 8, and / or the kit as claimed in claim 9, characterized in that, Used to detect Nipah virus.

Images