A dengue virus 4 detection primer based on multiplex RT-PCR and nucleic acid mass spectrometry method

Abstract

This invention discloses a primer and nucleic acid mass spectrometry method for detecting dengue virus genotypes 4 based on multiplex RT-PCR, belonging to the field of gene detection service technology. The primer set includes amplification primer pairs and extension primers. The amplification primer pairs include upstream and downstream primers specific to the poly genes of dengue virus types 1-4, with sequences shown in SEQ ID NO. 1-8, respectively. The extension primers include extension primers targeting specific sites of dengue virus types 1-4, with sequences shown in SEQ ID NO. 9-12, respectively. The nucleic acid mass spectrometry method of this invention includes: nucleic acid extraction, multiplex RT-PCR amplification and enrichment, SAP purification reaction, extension reaction, desalting treatment, and mass spectrometry detection. This invention not only utilizes the high throughput and high precision advantages of nucleic acid mass spectrometry technology, but also provides a new, efficient, accurate, and reliable dengue virus genotyping detection scheme through targeted primer design and systematic process integration, providing a powerful technical tool for the accurate diagnosis, epidemic monitoring, and prevention and control of dengue fever.

Description

Technical Field

[0001] This invention belongs to the field of gene detection service technology, and relates to a primer and nucleic acid mass spectrometry method for detecting dengue virus type 4 based on multiplex RT-PCR. Background Technology

[0002] dengue( Dengue fever DF is a mosquito mainly caused by Aedes aegypti (Fengella fasciata). Aedes aegypti ) and Aedes albopictus ( Aedes albopictus ( ) viral infectious diseases that spread.

[0003] Dengue fever is caused by dengue virus (Dengue virus) Dengue virus Dengue fever is caused by dengue virus (DENV) infection. The main clinical symptoms include fever, headache, rash, joint pain, and bleeding. Severe cases can lead to dengue hemorrhagic fever and dengue shock syndrome. Dengue virus belongs to the Flaviviridae family, is a single-stranded RNA virus, and is approximately 50 nm in diameter. The envelope protein E determines the virulence of the virus, while the non-structural protein NS1 is associated with severe illness. There are four serotypes of dengue virus (DENV-1–4). There is no cross-immunity between serotypes; reinfection with other serotypes can lead to antibody-dependent enhancement, resulting in severe dengue fever and even death.

[0004] Therefore, dengue fever surveillance and typing are of great significance in dengue-endemic areas: First, they can provide early warning of severe illness risk, and rapid typing can identify atypical infections, enabling timely intervention and reducing mortality. Second, they facilitate the formulation of precise prevention and control strategies. Different serotypes have different epidemic trends and transmissibility; typing results can guide regions to carry out targeted vector control, vaccination (targeting specific serotypes), and surveillance of high-risk populations. Third, they facilitate epidemiological tracing and surveillance. Typing can trace the source of imported cases, dynamically assess viral mutations, and provide a basis for cross-border joint prevention and control of the epidemic.

[0005] Currently, dengue virus detection and typing mainly rely on the following technologies: virus isolation and culture is the "gold standard" but is too time-consuming; RT-PCR has high sensitivity but is costly and technically demanding; antigen / antibody detection is simple to operate but is susceptible to cross-reactions; high-throughput sequencing can comprehensively analyze the genome but is expensive and data analysis is complex. None of these methods can achieve an optimal balance between detection speed, cost, ease of operation, and accurate typing.

[0006] In recent years, the combination of multiplex PCR and mass spectrometry (MS) has provided new insights into virus typing. For example, Chinese invention patent CN113913551A discloses a multiplex PCR-MS detection method and kit for dengue virus typing. This method aims to achieve viral genotyping and sample tracing by designing primer sets targeting multiple targets and single nucleotide polymorphism (SNP) typing probes. However, in pursuit of high coverage and tracing capabilities, this method employs as many as 11 primer pairs and introduces an internal reference gene for sample origin determination. This results in a complex primer system, a relatively lengthy experimental procedure, and data analysis still requiring professional interpretation. In applications aimed at rapid clinical diagnosis and large-scale screening, there is still room for improvement in terms of ease of operation, detection speed, and cost control.

[0007] On the other hand, fluorescent PCR typing, represented by CN113430304A, is another mainstream technology. This type of method relies on the difference in optical signals from fluorescent groups and the Tm value of melting curves for indirect interpretation. Its multiplexing capability is limited by the number of available fluorescence channels and spectral overlap interference, and the detection results are easily affected by fluorescence quenching and non-specific amplification signals, leading to uncertainty in the typing results. More importantly, fluorescent PCR and nucleic acid mass spectrometry have fundamental differences in their detection principles: the former belongs to the category of indirect detection based on optical signals, while the latter is based on the direct physical determination of the absolute molecular weight of single-base extension products. This fundamental difference in principle determines that the primer and probe design rules of fluorescent PCR (only considering basic parameters such as Tm value and GC content) cannot be directly applied to nucleic acid mass spectrometry detection systems. Nucleic acid mass spectrometry methods require dedicated primer design, which must additionally meet a series of targeted and stringent constraints, including control of the molecular weight range of amplification primers, precise arrangement of the molecular weights of extension primers and their products (minimum mass difference ≥16 Da), and no cross-interference between multiple primer pairs.

[0008] Therefore, there is an urgent need in this field for a novel dengue virus typing method that is specifically designed for a streamlined nucleic acid mass spectrometry platform. This method would overcome the dual bottlenecks of complex procedures in existing mass spectrometry protocols and limitations in the principles of existing fluorescence protocols, while ensuring high specificity and high sensitivity, and achieve the best balance between operational procedures, detection costs, and typing accuracy. Summary of the Invention

[0009] The purpose of this invention is to provide a primer and nucleic acid mass spectrometry method for detecting dengue virus serotype 4 based on multiplex RT-PCR, addressing the common challenge in existing molecular typing techniques of balancing detection throughput, speed, cost, and operational complexity, which fails to simultaneously meet the key technical requirements of high sensitivity, high specificity, and intuitive results for rapid clinical diagnosis. This invention utilizes primers specifically enriched against the dengue virus polygene and combines them with extension primers targeting serotype-specific sites to construct a highly efficient multiplex RT-PCR amplification and mass spectrometry detection system. This achieves rapid, one-time closed-tube detection from sample to typing results while maintaining the inherent advantages of high throughput and high precision of nucleic acid mass spectrometry, avoiding complex sequencing analysis and bioinformatics interpretation.

[0010] The technical solution adopted in this invention provides a primer set, the key feature of which is that the primer set includes amplification primer pairs and extension primers; the amplification primer pairs include upstream and downstream primers specific to the poly genes of dengue virus types 1 to 4, with sequences shown in SEQ ID NO. 1 to 8 respectively; the extension primers include extension primers targeting specific sites of dengue virus types 1 to 4, with sequences shown in SEQ ID NO. 9 to 12 respectively; the primer set can achieve specific amplification and typing identification of four dengue virus types, and is compatible with multiplex RT-PCR amplification systems.

[0011] A nucleic acid mass spectrometry method for dengue virus type 4 based on multiplex RT-PCR, the key features of which include the following steps: S1. Nucleic acid extraction: Extract nucleic acid from the sample, control the nucleic acid purity A260 / A280 to be 1.7-2.0, and the concentration to be ≥5ng / μL to obtain template nucleic acid; S2. Multiplex RT-PCR amplification and enrichment: Using the amplification primer pairs in the primer set above, prepare the enrichment reaction premix, mix by shaking, dispense into aliquots, add the template nucleic acid as the amplification template, and follow the preset enrichment reaction program, including UNG enzyme reaction, reverse transcription, pre-denaturation and cyclic amplification, to complete the enrichment and obtain the amplification product. S3. SAP purification reaction: Add SAP reaction premix to the above amplification product, and perform purification and enzyme inactivation according to the preset SAP reaction program to obtain the purified product. S4. Extension reaction: Using the extension primers in the primer set above, prepare an extension reaction premix and add the purified product above. Follow the preset extension reaction procedure, and complete the reaction through pre-denaturation, cyclic annealing extension and final extension to obtain the extension product. S5. Desalination treatment: Add water and desalination resin to the above extended product, stir and mix well, and take the supernatant to obtain the desalination supernatant. S6. Mass spectrometry detection: Spot the above desalted supernatant onto the nucleic acid mass spectrometry chip, dry it, and then run it on the instrument. Through software settings, mass axis calibration, and spectrum acquisition, the genotyping detection of dengue virus type 4 is completed.

[0012] Furthermore, in step S2, the premixed enrichment reaction solution is composed of the following per test: 7 μL of amplification reaction solution, 2 μL of amplification enzyme solution, 3 μL of amplification primer mixture, and 3 μL of water, for a total volume of 15 μL; in each test, the amount of template nucleic acid added is 5 μL, forming an enrichment reaction system with a total volume of 20 μL.

[0013] Furthermore, the preset enrichment reaction procedure in step S2 is as follows: At 37℃, the UNG enzyme reaction was carried out for 1 minute, and the cycle was repeated once. Reverse transcription at 50℃ for 5 min, repeat once; Pre-denaturation at 95℃ for 5 minutes, repeated once; Then, the amplification cycle was performed at 95°C for 10 seconds, followed by annealing at 58°C for 15 seconds and extension at 72°C for 20 seconds, for a total of 45 cycles. After cyclic amplification, the final extension was performed at 72℃ for 3 min, and the cycle was repeated once; the sample was then stored at 4℃.

[0014] Furthermore, in step S3, the SAP reaction premix solution is composed of the following per test: 1 μL of SAP reaction solution and 2 μL of SAP enzyme solution; after adding the amplification product, a purification reaction system with a total volume of 25 μL is formed.

[0015] Furthermore, the preset SAP reaction procedure in step S3 is as follows: Purify at 37℃ for 30 min, repeat once; Inactivate at 80℃ for 5 min, repeat once; store at 4℃.

[0016] Furthermore, in step S4, the premixed solution for the extension reaction consists of the following components per test: 2 μL of extension reaction solution, 2 μL of extension enzyme solution, 1.5 μL of extension primer mixture, and 1.5 μL of water, for a total volume of 7 μL. After adding the purified product, an extension reaction system with a total volume of 32 μL is formed.

[0017] Furthermore, the aforementioned pre-defined extended reaction procedure in step S4 is as follows: Pre-denaturation at 95℃ for 30 seconds, repeated once; Then, the process proceeds to a cyclic annealing and extension, followed by denaturation at 95°C for 5 seconds, and then to a sub-cycle. The sub-cycle consists of annealing at 52°C for 5 seconds and extension at 80°C for 5 seconds. The sub-cycle is repeated 5 times. The denaturation and sub-cycle processes are repeated a total of 40 times. At 72°C, the final extension was performed for 3 minutes, and the cycle was repeated once; then stored at 4°C.

[0018] Specifically, the mixing time in step S5 is 5 min to 10 min.

[0019] More specifically, step S6 includes: opening the MassMore Typer software and setting the project method table; opening the EX-Acquire software, calibrating the mass axis, and then acquiring the spectrum; and analyzing the results after the spectrum acquisition is completed.

[0020] Compared with the prior art, the present invention has the following advantages: First, this invention constructs a detection system based on a nucleic acid mass spectrometry technology platform, which fundamentally breaks through the inherent limitations of the fluorescence PCR method and achieves a leapfrog improvement in typing specificity, result certainty, and detection throughput.

[0021] Current fluorescent PCR typing methods rely on indirect interpretation based on the optical signal of the fluorescent group and the Tm value of the melting curve. Their detection capability is limited by the number of fluorescent channels and spectral overlap interference, with multiplex detection throughput typically not exceeding four levels. Furthermore, the results are easily affected by fluorescence quenching and non-specific amplification signals. More critically, for clinical samples with low viral loads, fluorescent PCR often falls into the gray zone due to amplification curve signal decay, failing to provide a clear positive or negative conclusion. This necessitates relying on the operator's subjective experience to determine whether retesting is necessary, which not only increases testing costs and reporting time but also introduces uncertainty into clinical decision-making.

[0022] The nucleic acid mass spectrometry technology employed in this invention is based on the principle of matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. It achieves genotyping by directly determining the absolute molecular weight of specific single-base extension products. This revolutionary detection principle brings three fundamental advantages: First, multiplex detection is no longer limited by the number of fluorescence channels; a single tube can simultaneously complete genotyping of four serotypes, and this can be further expanded to more than 20, reserving ample space for multi-pathogen joint detection. Second, the results are expressed as the presence or absence of characteristic peaks at specific mass-to-charge ratio positions in the mass spectrum, with objective and clear judgment criteria. There is no need to set threshold lines or rely on manual experience to interpret the curve morphology, resulting in a high degree of automation. Third, clinical sample validation shows that even for low viral load samples with high Ct values ​​detected by fluorescent PCR, located in the gray zone, and requiring retesting for confirmation, this method can still output clear, single extension product peaks, completing positive interpretation and accurate genotyping in one step. This significantly reduces retesting costs and shortens report turnaround time, providing a valuable window for early clinical diagnosis and timely intervention. This advantage is something that existing fluorescent PCR methods cannot achieve at the principle level.

[0023] Second, the present invention lays the core foundation for realizing the above-mentioned technical advantages through a simplified and efficient specific primer set design.

[0024] Unlike existing approaches that employ complex primer sets to achieve high coverage, this invention precisely targets highly conserved polygenic regions and designs core amplification and extension primers for specific SNP sites of four serotypes. More importantly, the design of the extension primers adheres to the stringent constraints unique to nucleic acid mass spectrometry platforms: through precise calculations, the molecular weights of the four extension primers and their extension products are ensured to be non-overlapping in the mass spectrum, with the minimum mass difference strictly controlled above 16 Da; simultaneously, targeted sequence modifications and molecular weight checks are performed on the 5' ends of the amplification primers to ensure their molecular weights exceed the detection window, resulting in a clear and interference-free red primer peak and blue product peak pattern on the spectrum. This multi-dimensional and strongly constrained design logic cannot be achieved using conventional primer design software and fluorescent PCR design experience.

[0025] Third, through process optimization, this invention achieves deep and efficient integration of multiplex RT-PCR and nucleic acid mass spectrometry technologies, and exhibits excellent specificity and anti-interference capabilities.

[0026] In the method specificity validation, the primer set used showed high specificity for dengue virus type 1 and no cross-reactivity with the other three serotypes and other common vector-borne infectious disease pathogens (such as chikungunya virus, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, etc.).

[0027] The underlying reason for this experimental result lies in the fact that the single-base extension enzyme used in this invention has a much higher fidelity than the Taq enzyme used in fluorescent PCR. The 3' end of the extension primer must perfectly match the template for the extension reaction to occur. In contrast, even if there are a few mismatches between the fluorescent PCR probe and the target sequence's 3' end, hybridization and fluorescence signals may still occur, leading to a risk of non-specific signals when dealing with closely related pathogens with homologous sequences. This invention fundamentally avoids such false-positive interference at the enzymatic level, resulting in higher typing accuracy.

[0028] Furthermore, this invention introduces an UNG enzyme anti-contamination step, forming a closed-loop, automated operation process from nucleic acid amplification to mass spectrometry typing, which significantly improves the stability and reproducibility of the method.

[0029] Fourth, the results of this invention are intuitive and efficient to interpret, reducing the reliance on complex bioinformatics analysis and making it highly practical.

[0030] The results of this invention are directly reflected in the presence and location of characteristic mass peaks in the mass spectrum. Each serotype corresponds to a unique and expected characteristic peak, making the judgment criteria objective and clear, greatly simplifying the analysis process, and making it more suitable for rapid diagnosis and large-scale applications. Clinical sample validation showed that its detection results are highly consistent with current fluorescent RT-PCR methods, and it performed well in samples with low viral loads, demonstrating excellent clinical detection efficacy and translational potential.

[0031] In summary, this invention not only leverages the high throughput and high precision advantages of nucleic acid mass spectrometry, but also provides a novel, efficient, accurate, and reliable dengue virus typing detection solution through targeted primer design and systematic workflow integration. In particular, this invention effectively addresses the core clinical pain points of existing fluorescent PCR methods, such as ambiguous interpretations and reliance on retesting in cases with low viral loads, providing a powerful technical tool for the accurate diagnosis, epidemic monitoring, and prevention of dengue fever. Attached Figure Description

[0032] Figure 1 This is a nucleic acid mass spectrum of a dengue virus type I (DENV-1) positive sample; Figure 2 This is a mass spectrum of nucleic acid from a dengue virus type II (DENV-2) positive sample; Figure 3 This is a mass spectrum of nucleic acid from a dengue virus type III (DENV-3) positive sample; Figure 4 This is a mass spectrum of nucleic acid from a dengue virus type IV (DENV-4) positive sample; Figure 5 This is the mass spectrum of dengue virus type I (DENV-1) from sample number 3; Figure 6 This is the mass spectrum of dengue virus type II (DENV-2) from sample number 13; Figure 7 This is the mass spectrum of dengue virus type III (DENV-3) from sample number 20. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] Unless otherwise specified in the examples, the procedures can be followed according to conventional conditions; unless the manufacturers of the reagents or instruments used are specified, they are all conventional products that can be purchased commercially.

[0039] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0040] Example 1 This embodiment provides a primer set for detecting four types of dengue virus and a nucleic acid mass spectrometry method, as detailed below: S1. Nucleic acid extraction: Select the appropriate extraction kit according to the sample type and experimental project, and extract nucleic acid from the serum sample to be tested; determine the purity and concentration of nucleic acid: A260 / A280 = 1.7~2.0; concentration ≥5ng / uL.

[0041] S2, multiplex RT-PCR amplification and enrichment: S21. Design of amplification primer pairs: The enrichment primers designed for dengue virus types 1 to 4 (DENV-1 to 4) in this invention have sequences shown in Table 1 as SEQ ID NO.1 to 8.

[0042] Table 1: Amplification primer sequences for DENV-1 to DENV-4

[0043] The primer set of this invention was designed in accordance with the principles of systematicity, specificity, and multiple compatibility.

[0044] Regarding target gene selection, all primer pairs specifically target the polygene of dengue virus, namely the gene region encoding viral RNA-dependent RNA polymerase. Comparison with a large number of viral genome sequences revealed that this region is crucial for maintaining viral replication function and therefore exhibits high sequence conservation within the same serotype. This ensures the primer pairs' broad capture capability of different circulating strains within each serotype, laying the foundation for high-sensitivity detection. Simultaneously, the sequences in this region of different serotypes exhibit stable and sufficient differences, providing feasibility for designing serotype-specific primers, thus fundamentally guaranteeing the specificity of genotyping detection.

[0045] To address the natural genetic variations within viral populations, the primer set design of this invention carefully incorporates degenerate bases at key sites. For example, the downstream primer for DENV-1 (SEQ ID NO.5) uses "Y" to represent pyrimidine, and the upstream primer for DENV-3 (SEQ ID NO.3) uses "R" to represent purine. This design actively accommodates known single nucleotide polymorphisms, significantly improving primer coverage for different genotypes or variants within the same serotype without sacrificing the stringency of primer-target sequence binding, effectively reducing the risk of false negatives due to minor mutations in the target sequence.

[0046] Furthermore, this invention also finely controls the length of the amplification primer sequences. The target fragments amplified by the four primer pairs were designed to be 131bp, 78bp, 91bp, and 108bp in length, respectively, all of which are short fragment amplifications. In the RT-PCR process, short fragments not only have higher amplification efficiency and faster speed, especially suitable for RNA templates that may be partially degraded in clinical samples, but also ensure that all amplicons are of similar length, thus ensuring a more balanced amplification kinetics among the targets in subsequent multiplex PCR reactions in the same tube. This avoids amplification competition and bias caused by excessive differences in product length, thereby ensuring the quantitative reliability and reproducibility of multiplex detection.

[0047] Regarding amplicon design, the complete polygene sequences based on DENV-1 to 4 have been published in public databases. Based on the primer sequences and amplicon lengths of each serotype disclosed in this application, and in conjunction with the aforementioned known reference sequences, those skilled in the art can directly and without doubt determine the complete sequence of each serotype amplicon.

[0048] S22, Enrichment reaction process: Prepare the enrichment reaction premix according to Table 2.

[0049] Table 2: Formulation of enrichment reaction premix

[0050] Table 2 lists the amplification reaction solution, amplification enzyme solution, and amplification primer mixture as standard reagents necessary for performing multiplex RT-PCR amplification reactions. The amplification reaction solution provides the necessary buffer system, magnesium ions, and substrate dNTPs; the amplification enzyme solution provides a hot-start DNA polymerase with reverse transcription function; and the amplification primer mixture contains specific amplification primers as shown in SEQ ID NO. 1–8 (primer concentrations are shown in Table 1). Those skilled in the art can select appropriate commercially available multiplex RT-PCR premixed kits or prepare equivalent standard reagents according to their testing needs.

[0051] Prepare the reagents in EP tubes according to Table 2. Shake the prepared enrichment reaction premixed solution to mix well. Dispense 15 μL / test into PCR eight-tube strips. Add 5 μL of template to each test tube to obtain the enrichment reaction system. Tightly cap the tubes and centrifuge briefly.

[0052] Set the enrichment program according to Table 3, perform PCR amplification, and obtain the amplification product.

[0053] Table 3: Enrichment Reaction Procedure

[0054] S3, SAP (shrimp alkaline phosphatase) purification reaction: Prepare the SAP reaction premix according to Table 4.

[0055] The SAP reaction solution and SAP enzyme solution are standard reagents necessary for PCR product purification. The SAP reaction solution provides a buffer system for shrimp alkaline phosphatase, while the SAP enzyme solution provides shrimp alkaline phosphatase to degrade residual dNTPs in the system. Those skilled in the art can select appropriate commercially available SAP purification reagents or prepare equivalent standard reagents according to standard molecular experimental procedures.

[0056] Table 4: Formulation of SAP Reaction Premix

[0057] Prepare the reagents in EP tubes according to the dosages shown in Table 4.

[0058] If the number of sample reactions is 10, the amount of each component added is 10 μL and 20 μL respectively. After shaking and centrifuging, the prepared SAP reaction premix solution is shaken and mixed well. It is then aliquoted into the eight-tube amplification product obtained in step S2 at a rate of 3 μL / test, the tube caps are tightened, and the tubes are centrifuged briefly.

[0059] Following the SAP reaction program set in Table 5, the purification reaction was carried out on the machine to obtain the purified product.

[0060] Table 5: SAP Reaction Procedure

[0061] S4, Extended reaction: S41. Design of extension primers: The extension primers designed for dengue virus types 1 to 4 (DENV-1 to 4) in this invention have sequences shown in SEQ ID NO. 9 to 12 in Table 6.

[0062] Table 6: Extension primer sequences for DENV-1 to 4

[0063] The core design principle of the extended primers (sequences shown in SEQ ID NO. 9-12) designed in this invention is to achieve direct and clear typing based on mass spectrometry peaks.

[0064] The binding site of the extension primer of this invention is located upstream of the type-specific single nucleotide site within the corresponding serotype enrichment product, and the base immediately adjacent to its 3' end has inherent differences between different types. In the single-base extension reaction, by adding a specific dideoxynucleotide (ddNTP), each type produces only one extension product with a fixed molecular weight.

[0065] In the design of extension primers, this invention overcomes challenges far exceeding those of conventional PCR primer design. First, through extensive sequence alignment, specific and conserved SNP sites on the poly genes of the four serotypes were precisely anchored, and the 3' ends of the extension primers were designed to be immediately upstream of these SNP sites. Second, to ensure the accuracy of mass spectrometry typing, precise calculations were performed on the arrangement of eight molecular weights, including the four extension primers themselves and the extension products after adding a single ddNTP. The design required that each characteristic peak be completely separated on the mass spectrum (m / z 4500-9000), with the minimum molecular weight difference strictly controlled above the effective mass spectrometry resolution of ≥16 Da to prevent misinterpretation due to peak ligation.

[0066] Meanwhile, this invention has carried out targeted sequence modifications and molecular weight checks at the location of the extension primers and the 5' end of the amplification primers to ensure that the molecular weight of all primers jumps out of the detection window, thereby forming a clear and interference-free "red primer peak" and "blue product peak" interpretation pattern on the spectrum.

[0067] This invention, through precise calculation and optimization, ensures sufficient differences in the molecular weight (mass-to-charge ratio) of the four extension primers and their final extension products. This results in well-defined, non-interfering characteristic peaks in the mass spectrum, enabling intuitive spectral interpretation and automated typing. The concentrations of each extension primer shown in Table 6 are the result of experimental optimization, balancing the extension efficiency of different types in the multiplex extension reaction system. This compensates for potential differences in reaction kinetics between different primer-template complexes, ensuring balanced mass spectrometry signal intensity across the four channels and avoiding missed or misjudged typing due to uneven signal strength.

[0068] S42. Extended reaction process: Prepare the extension reaction premix according to Table 7.

[0069] Table 7: Formulation of the extension reaction premix

[0070] The extension reaction solution, extension enzyme solution, and extension primer mixture in Table 7 are standard reagents necessary for performing single-base extension reactions. The extension reaction solution provides the buffer system and substrate (including four ddNTPs) required for the reaction; the extension enzyme solution provides a thermostable DNA polymerase; and the extension primer mixture contains extension primers as shown in SEQ ID NO. 9–12 (primer concentrations are shown in Table 6). Those skilled in the art can select appropriate commercially available kits or prepare equivalent standard reagents according to the specific mass spectrometry platform used.

[0071] Prepare the components according to the dosage shown in Table 7 in EP tubes. If the number of sample reactions is 10, the amount of each component added is 20 μL, 20 μL, 15 μL, and 15 μL, respectively. After shaking and centrifuging, shake the prepared extension reaction premixed solution to mix well, and add 7 μL / test to the purified reaction product obtained in step S3.

[0072] Design the extension reaction procedure according to Table 8 and carry out the extension reaction on the machine.

[0073] Table 8: Extended Reaction Procedure

[0074] The extension reaction of this invention is based on the single-base extension principle. Extension primers (SEQ ID NO. 9–12) anneal to the purified PCR product at their corresponding serotype-specific sites. In subsequent reactions, DNA polymerase terminates the reaction by incorporating only one complementary ddNTP at the 3' end of the extension primer, according to the template sequence.

[0075] Because different types of target bases are different, the types and molecular weights of ddNTPs incorporated are also different, resulting in type-specific extended products with mass numbers for mass spectrometry detection and typing.

[0076] S5. Desalination treatment: Add 40 μL of experimental pure water to the eight-tube set; then add 30 mg of desalination resin, and mix thoroughly by inverting the tube for 5 to 10 minutes.

[0077] S6. Mass spectrometry detection: S61, Chip Spotting: 20 nL of the desalted product supernatant was transferred to cover the corresponding detection area of ​​the nucleic acid mass spectrometry chip, dried, and then used for detection. S62, On-the-job training: Open the MassMore Typer software and set the project method table; Open the EX-Acquire software, calibrate the mass axis, and then acquire the spectrum. Once the spectrum acquisition is complete, the results can be analyzed.

[0078] S63. Result Interpretation: The results interpretation of this invention is based on the presence and positional characteristics of extended primer and extended product peaks in nucleic acid mass spectrometry. It eliminates the need for complex sequence alignment or bioinformatics analysis; positive determination and serotyping can be completed directly through the position of characteristic peaks. The specific interpretation rules are as follows: (1) Interpretation of DENV-1 type mass spectra (e.g.) Figure 1 (as shown) The extension primer peak (red) is located at a mass-to-charge ratio (m / z) of 5739.8 Da, corresponding to the molecular weight of the DENV-1 extension primer (SEQ ID NO. 9). This peak is the background signal of the reaction system, indicating that the extension primer has been added to the reaction normally, and is a basic indicator of the system's effectiveness.

[0079] The extended product peak (blue): located at m / z 6011 Da, is a characteristic peak unique to DENV-1 positive samples. This peak is the product of the extension primer incorporating a specific ddNTP under the guidance of the DENV-1 template. The molecular weight increment (6011-5739.8=271.2 Da) is completely matched with the molecular weight of the incorporated ddNTP.

[0080] Judgment criteria: If a sample shows a clear blue product peak at 6011 Da (signal intensity ≥ threshold) and a red primer peak is present, it is judged as DENV-1 positive; if only a red primer peak is present, it is judged as DENV-1 negative.

[0081] (2) Interpretation of DENV-2 type mass spectra (e.g.) Figure 2 (as shown) The extension primer peak (red) is located at m / z 4914.3 Da, corresponding to the molecular weight of the DENV-2 extension primer (SEQ ID NO.10).

[0082] Extended product peak (blue): located at m / z 5201.5 Da, the molecular weight increment (5201.5-4914.3=287.2 Da) is completely consistent with the DENV-2 specific ddNTP incorporation.

[0083] Judgment criteria: If a blue product peak appears at 5201.5 Da, the sample is judged to be DENV-2 positive; if only a red primer peak is present, the sample is DENV-2 negative.

[0084] (3) Interpretation of DENV-3 mass spectra (e.g.) Figure 3 (as shown) The extension primer peak (red) is located at m / z 7343.8 Da, corresponding to the molecular weight of the DENV-3 extension primer (SEQ ID NO.11).

[0085] Extended product peak (blue): located at m / z 7591 Da, the molecular weight increase (7591-7343.8=247.2 Da) is consistent with the DENV-3 specific ddNTP incorporation characteristics.

[0086] Judgment criteria: If a blue product peak appears at 7591 Da, the sample is judged to be DENV-3 positive; if only a red primer peak is present, the sample is DENV-3 negative.

[0087] (4) Interpretation of DENV-4 type nucleic acid mass spectra (e.g.) Figure 4 (as shown) The extension primer peak (red) is located at m / z 7404.9 Da, corresponding to the molecular weight of the DENV-4 extension primer (SEQ ID NO.12).

[0088] Extended product peak (blue): located at m / z 7731.6 Da, the molecular weight increment (7731.6-7404.9=326.7 Da) is a perfect match for the DENV-4 specific ddNTP incorporation.

[0089] Judgment criteria: If a blue product peak appears at 7731.6 Da, the sample is judged to be DENV-4 positive; if only a red primer peak is present, the sample is DENV-4 negative.

[0090] By jointly analyzing the peak positions of the four spectra, simultaneous typing of four dengue virus types can be achieved: Single-type infection: The blue product peak of the corresponding type appears only in one spectrum, while other spectra only show red primer peaks, which is determined to be a single infection of that type.

[0091] Mixed infection: If blue product peaks appear in two or more spectra, it is determined to be a mixed infection of the corresponding type (such as mixed infection of DENV-1 and DENV-2).

[0092] Negative determination: All spectra showed only red primer peaks and no blue product peaks, indicating a negative result for dengue virus.

[0093] Example 2 This embodiment verifies the compliance rate of the method of the present invention, that is, by using 12 copies of DENV-1, DENV-2, DENV-3, and DENV-4 types respectively. 5 The standard was tested in copies / mL, and negative samples were tested using NC (pure water) as the method compliance rate. The entire process was carried out according to the instructions of the nucleic acid purification kit. The test results were recorded and compared with the theoretical types. The negative compliance rate and positive compliance rate after comparison were recorded. The results are shown in Tables 9 and 10.

[0094] Table 9: Experimental Data on the Concordance Rate Test for Negative and Positive Sexes

[0095] Table 10: Experimental Data on the Concordance Rate Test for Negative and Positive Sexes (Part Two)

[0096] As shown in Tables 9 and 10, all 12 DENV-1 standards were positive in the DenV-1 detection channel, all 12 DENV-2 standards were positive in the DenV-2 detection channel, all 12 DENV-3 standards were positive in the DenV-3 detection channel, and all 12 DENV-4 standards were positive in the DenV-4 detection channel. The control group, with NC (pure water) as the negative sample, was negative. The positive and negative concordance rates of this method were both 100%, which can accurately distinguish the four serotypes of dengue virus.

[0097] Example 3 This embodiment aims to verify the consistency between the method of the present invention and current conventional methods through clinical samples, and to evaluate its clinical detection efficacy. Thirty suspected clinical samples from patients (samples 1 to 30) were selected and tested according to the method of Embodiment 1 of the present invention. The concordance rate of the method of the present invention was calculated using fluorescent RT-PCR results as the reference standard. The results are shown in Table 11, and the test chromatograms of some samples are shown in [Table 11]. Figures 5-7 .

[0098] The detection protocol for fluorescent RT-PCR in this embodiment is implemented as follows: The detection was performed using the commercially available "Dengue Virus Type I / II / III / IV Typing Kit (Fluorescent PCR Method)" manufactured by Shanghai ZJ Bio-Tech Co., Ltd.

[0099] Specifically, it includes: After extracting RNA from the sample to be tested, a one-step RT-PCR reaction system was prepared using specific primers for dengue virus types 1-4 and TaqMan fluorescent probes.

[0100] The total volume of the reaction system was 25 μL, containing 19 μL of dengue virus typing fluorescent PCR mixture, 1 μL of RT-PCR enzyme, and 5 μL of nucleic acid from the sample to be tested.

[0101] The reaction program was as follows: 50℃, 5 min; 95℃, 3 min; 95℃, 5 s; 60℃, 30 s, for a total of 45 cycles; fluorescence signals were collected at 60℃.

[0102] Results are determined as follows: a Ct value ≤ 41 with a typical S-shaped amplification curve is considered positive; a Ct value of 41–45 or an atypical amplification curve is considered gray and requires retesting for confirmation.

[0103] Table 11: Test Results of Clinical Suspected Samples

[0104] As shown in Table 11, in 30 clinical samples, the nucleic acid mass spectrometry detection results of this invention were completely consistent with the fluorescence RT-PCR results, with a total concordance rate of 100%. Among them, the detection results of DENV-1, DENV-2, and DENV-3 positive samples were all one-to-one.

[0105] Of particular note are samples 2, 4, 10, and 19, whose CT values ​​for fluorescent RT-PCR were 36.76, 37.21, 38.38, and 35.38, respectively, classifying them as samples with low viral load (CT value > 35). The method of this invention was still able to successfully detect and correctly genotype these samples, indicating that the method of this invention has clinical sensitivity comparable to, or even potentially superior to, fluorescent RT-PCR.

[0106] Furthermore, the method of this invention can simultaneously complete the typing detection of four serotypes in a single tube reaction, and is compatible with internal reference, quality control, and other pathogen targets (expandable to more than 20), while the fluorescent RT-PCR method is limited by the number of fluorescent channels (usually ≤4), and the typing of the four types requires multiple tubes / multiple channels to be detected separately.

[0107] Note: The samples collected in this clinical validation were all from the current circulating strains and did not include DENV-4 positive cases. This situation is consistent with the epidemiological characteristics of the periodic epidemic of specific dengue serotypes and does not affect the evaluation of the detection performance of the method of this invention.

[0108] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer set, characterized in that, The primer set includes amplification primer pairs and extension primers; the amplification primer pairs include upstream and downstream primers specific to the polygenes of dengue virus types 1-4, with sequences shown in SEQ ID NO. 1-8, respectively; the extension primers include extension primers targeting specific sites of dengue virus types 1-4, with sequences shown in SEQ ID NO. 9-12, respectively; the primer set can achieve specific amplification and typing identification of four dengue virus types, and is compatible with multiplex RT-PCR amplification systems.

2. A nucleic acid mass spectrometry method for dengue virus type 4 based on multiplex RT-PCR, characterized in that, Includes the following steps: S1. Nucleic acid extraction: Extract nucleic acid from the sample, control the nucleic acid purity A260 / A280 to be 1.7-2.0, and the concentration to be ≥5ng / μL to obtain template nucleic acid; S2. Multiplex RT-PCR amplification and enrichment: Using the amplification primer pairs in the primer set as described in claim 1, prepare an enrichment reaction premix, mix thoroughly by shaking, dispense into aliquots, add the template nucleic acid as the amplification template, and follow the preset enrichment reaction program, including UNG enzyme reaction, reverse transcription, pre-denaturation and cyclic amplification, to complete the enrichment and obtain the amplification product. S3. SAP purification reaction: Add SAP reaction premix to the amplification product, and perform purification and enzyme inactivation according to the preset SAP reaction program to obtain the purified product. S4. Extension reaction: Using the extension primers in the primer set as described in claim 1, prepare an extension reaction premix and add it to the purified product. Follow the preset extension reaction procedure to complete the reaction through pre-denaturation, cyclic annealing extension and final extension to obtain the extension product. S5. Desalination treatment: Add water and desalination resin to the extended product, stir and mix well, and take the supernatant to obtain the desalination supernatant. S6. Mass spectrometry detection: The desalted supernatant is spotted onto the nucleic acid mass spectrometry chip, dried, and then loaded onto the instrument. Through software settings, mass axis calibration, and spectrum acquisition, the genotyping detection of dengue virus type 4 is completed.

3. The nucleic acid mass spectrometry method according to claim 2, characterized in that, The enrichment reaction premix in step S2 consists of the following components per test: 7 μL of amplification reaction solution, 2 μL of amplification enzyme solution, 3 μL of amplification primer mixture, and 3 μL of water, for a total volume of 15 μL. In each test, the amount of template nucleic acid added is 5 μL, which together form an enrichment reaction system with a total volume of 20 μL.

4. The nucleic acid mass spectrometry method according to claim 2, characterized in that, The preset enrichment reaction procedure mentioned in step S2 is as follows: At 37℃, the UNG enzyme reaction was carried out for 1 minute, and the cycle was repeated once. Reverse transcription at 50℃ for 5 min, repeat once; Pre-denaturation at 95℃ for 5 minutes, repeated once; Then, the amplification cycle was performed at 95°C for 10 seconds, followed by annealing at 58°C for 15 seconds and extension at 72°C for 20 seconds, for a total of 45 cycles. After cyclic amplification, the final extension was performed at 72℃ for 3 min, and the cycle was repeated once; the sample was then stored at 4℃.

5. The nucleic acid mass spectrometry method according to claim 2, characterized in that, The SAP reaction premix in step S3 consists of the following components per test: 1 μL of SAP reaction solution and 2 μL of SAP enzyme solution; after adding the amplification product, a purification reaction system with a total volume of 25 μL is formed.

6. The nucleic acid mass spectrometry method according to claim 2, characterized in that, The preset SAP reaction procedure mentioned in step S3 is as follows: Purify at 37℃ for 30 min, repeat once; Inactivate at 80℃ for 5 min, repeat once; store at 4℃.

7. The nucleic acid mass spectrometry method according to claim 2, characterized in that, The premixed solution for the extension reaction in step S4 consists of the following components per test: 2 μL of extension reaction solution, 2 μL of extension enzyme solution, 1.5 μL of extension primer mixture, and 1.5 μL of water, for a total volume of 7 μL. After adding the purified product, an extension reaction system with a total volume of 32 μL is formed.

8. The nucleic acid mass spectrometry method according to claim 2, characterized in that, The preset extension reaction procedure mentioned in step S4 is as follows: Pre-denaturation at 95℃ for 30 seconds, repeated once; The process then proceeds to a cyclic annealing and extension, followed by denaturation at 95°C for 5 seconds, and then to a sub-cycle. The sub-cycle consists of annealing at 52°C for 5 seconds and extension at 80°C for 5 seconds. The sub-cycle is repeated 5 times. The denaturation and sub-cycle processes are repeated a total of 40 times. At 72°C, the final extension was performed for 3 minutes, and the cycle was repeated once; then stored at 4°C.

9. The nucleic acid mass spectrometry method according to claim 2, characterized in that, The mixing time described in step S5 is 5 min to 10 min.

10. The nucleic acid mass spectrometry method according to claim 2, characterized in that, Step S6 specifically includes: opening the MassMoreTyper software and setting the project method table; opening the EX-Acquire software, calibrating the mass axis, and then acquiring the spectrum; and analyzing the results after the spectrum acquisition is completed.

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