Multiplex amplification method, system and applications

Abstract

The application is suitable for the biotechnology field, and provides a multiplex amplification method and system and application, wherein the multiplex amplification method comprises the following steps: linear amplification of a target template by using an upstream primer mixture designed for multiple target regions to obtain a first product by purification; the 5' end of the upstream primer contains a universal primer sequence, and the 3' end contains a specific target recognition sequence; linear amplification of the first product by using a downstream primer mixture designed for multiple target regions to obtain a second product by purification; the 5' end of the downstream primer contains a universal primer sequence, and the 3' end contains a specific target recognition sequence; and unified exponential amplification of the second product by using a universal primer. The application innovatively adopts a specific multi-target molecular marker combined with a universal primer amplification strategy to establish a brand-new multi-target DNA fragment amplification technology system, and high uniformity, high specificity and high-throughput multi-target amplification can be realized.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a multiplex amplification method, system, and application. Background Technology

[0002] Multiplex PCR is a molecular biology technique that uses multiple pairs of primers to amplify multiple target DNA fragments simultaneously in a single reaction system. It has become a core technology tool in fields such as molecular diagnostics, genetic disease screening, pathogen detection, and genotyping.

[0003] Traditional multiplex PCR works by mixing all specific primers designed for different targets in the same reaction system and simultaneously amplifying all target fragments through PCR thermal cycling (denaturation-annealing-extension). The technical process includes: first, designing a pair of specific primers for each target, ensuring that all primers have similar Tm values ​​to avoid primer dimer formation; second, mixing all primer pairs at optimized concentrations, adding DNA template, dNTPs, buffer, and DNA polymerase; then amplifying through thermal cycling; and finally detecting the products by gel electrophoresis, capillary electrophoresis, or high-throughput sequencing.

[0004] Existing multiplex PCR techniques mainly include the following forms: The most basic traditional multiplex PCR directly adds all target-specific primer pairs to the reaction system for simultaneous amplification. However, when the number of targets exceeds 10, primer interactions increase significantly, and the optimization difficulty increases exponentially. Therefore, in practical applications, the number of targets amplified simultaneously is usually limited to 10-20. Nested multiplex PCR improves specificity and sensitivity through two rounds of amplification. The first round uses outer primers for preliminary amplification of multiple targets, and the second round uses inner primers located inside the product for specific amplification. However, this method is cumbersome, has a high risk of cross-contamination, and is not conducive to clinical application. Multiplex ligation-dependent probe amplification (MLPA) is another method. The first technology uses two half-probes to hybridize to adjacent target sequences, ligates the probes using ligase, and then amplifies them with universal primers. It can simultaneously detect 40-50 targets, but requires an additional ligase reaction step, resulting in higher costs and stricter DNA quality requirements. Multiplex PCR combined with high-throughput sequencing combines multiplex amplification with next-generation sequencing, enabling simultaneous detection of hundreds of target sites, but still faces challenges such as amplification bias, complex primer optimization, and high costs. Furthermore, targeted capture sequencing uses biotin-labeled probes to capture target regions before sequencing, covering a larger genomic region, but its cost is significantly higher than PCR methods, and it requires high throughput, making it unsuitable for small-scale, rapid clinical testing needs. Product development focused on specific scenarios, such as pathogen detection and tumor gene sequencing kits, requires independent optimization, resulting in high development costs.

[0005] In summary, although multiplex PCR technology has significant theoretical advantages, traditional multiplex PCR faces numerous technical bottlenecks in practical applications, severely limiting its widespread use in high-throughput detection, clinical diagnosis, and basic research. Furthermore, existing technologies are all improvements within the traditional framework and have failed to overcome the fundamental limitation of multiple primer competition. Specifically, existing improved technologies still have the following main drawbacks: when multiple primer pairs coexist in the same reaction system, various non-productive interactions can form between primers: the formation of primer dimers consumes reaction components and generates a large number of non-specific products, forming low-molecular-weight bands in gel electrophoresis that interfere with result interpretation, and consuming valuable sequencing reads in sequencing; cross-hybridization and mismatches between primers lead to the amplification of non-target sequences, reducing detection specificity; non-specific binding of primers to non-target sites is difficult to completely avoid, especially when the genomic DNA content is high or the target sequence similarity is high. More importantly, due to inherent differences in GC content, length, and secondary structure, different target fragments exhibit significant differences in amplification efficiency. Strongly competitive primer pairs (high Tm value, high GC content) preferentially amplify and consume reaction components, inhibiting the amplification efficiency of weakly competitive primer pairs. This ultimately leads to over-amplification of some targets and under-amplification of others. This severe amplification heterogeneity and nonspecificity not only reduces the accuracy and sensitivity of detection but also makes traditional multiplex PCR difficult to apply to quantitative detection and the detection of low-abundance targets. Summary of the Invention

[0006] The purpose of this invention is to provide a multiplex amplification method that aims to solve the problems mentioned in the background art.

[0007] To address the above problems, this invention provides a multiplex amplification method, comprising the following steps:

[0008] Upstream primer labeling reaction: A mixture of upstream primers designed for multiple target regions is used to linearly amplify the target template and purify it to obtain the first product; the 5' end of the upstream primer contains a universal primer sequence and the 3' end contains a specific target recognition sequence.

[0009] Downstream primer labeling reaction: The first product is linearly amplified using a mixture of downstream primers designed for multiple target regions, and the second product is purified; the 5' end of the downstream primers contains a universal primer sequence, and the 3' end contains a specific target recognition sequence.

[0010] Universal primer homogenization amplification: The second product is uniformly amplified exponentially using universal primers designed based on the universal primer sequence.

[0011] Furthermore, the universal primer sequence has no specific binding site to the target template, and the primer design is optimized to reduce secondary structures and primer dimer formation.

[0012] Furthermore, the Tm value of the universal primer sequence is ≥60℃.

[0013] Furthermore, the universal primer sequence is shown in the sequence listing SEQ ID NO:1.

[0014] Furthermore, the steps of linearly amplifying the target template and purifying it to obtain the first product using a mixture of upstream primers designed for multiple target regions specifically include:

[0015] In a PCR reaction system containing the target template, a mixture of upstream primers designed for multiple target regions is added, and a linear amplification reaction is performed for 1-5 cycles. Universal primer sequences are labeled upstream of the target region. After the reaction, the unreacted upstream primer mixture is removed by purification with magnetic beads to obtain the first product.

[0016] Furthermore, the step of linearly amplifying the first product and purifying it to obtain the second product using a mixture of downstream primers designed for multiple target regions specifically includes:

[0017] In a PCR reaction system including the first product, a mixture of downstream primers designed for multiple target regions is added, and a linear amplification reaction is performed for 1-5 cycles. Universal primer sequences are labeled downstream of the target regions, and the extension products cover the upstream primer regions, so that both ends of the target regions are labeled with universal primer sequences. After the reaction is completed, the unreacted downstream primer mixture is removed by purification with magnetic beads to obtain the second product.

[0018] Furthermore, the above-mentioned multiplex amplification method also includes the following steps:

[0019] By combining shingled amplification, amplicon sequencing, and de novo assembly strategies, complete coverage of long target regions can be achieved, and complex structural variations can be detected through short-read sequencing.

[0020] Another object of the present invention is to provide a multiplex amplification system for implementing the above-described multiplex amplification method, comprising DNA polymerase, dNTPs and reaction buffer, and further comprising one or more upstream and downstream primers; wherein the 5' end of the upstream primer contains a universal primer sequence and the 3' end contains a specific target recognition sequence; wherein the 5' end of the downstream primer contains a universal primer sequence and the 3' end contains a specific target recognition sequence.

[0021] Another objective of this invention is to provide an application of the above-mentioned multiplex amplification method or multiplex amplification system in the preparation of multipathogen detection kits, which can achieve high-throughput, high-sensitivity and field-operable molecular diagnostics.

[0022] Another objective of this invention is to provide an application of the above-mentioned multiplex amplification method or multiplex amplification system in the preparation of a genetic disease gene mutation detection kit; the detection includes full coverage of the full gene length and accurate identification of mutation sites.

[0023] Another objective of this invention is to provide the application of the above-mentioned multiplex amplification method or multiplex amplification system in the preparation of multi-gene joint screening kits, so as to realize the parallel detection and mutation analysis of multiple disease-related genes.

[0024] The multiplex amplification method provided by this invention focuses on the efficient amplification of multiple target fragments, aiming to achieve uniform, specific, and reproducible amplification of multiple DNA fragments. By innovatively employing a combination of specific multi-target molecular markers and universal primer amplification strategies, a novel multi-target DNA fragment amplification technology system is established. This system can achieve high uniformity, high specificity, and high throughput multi-target amplification, providing a simple, accurate, economical, and reliable technical solution for clinical and scientific research applications such as genetic disease detection and pathogen detection. Attached Figure Description

[0025] Figure 1 This is a schematic flowchart of the multiplex amplification method provided in an embodiment of the present invention.

[0026] Figure 2 This is a flowchart illustrating the application verification of the multiplex amplification method provided in this embodiment of the invention.

[0027] Figure 3 This is a comparison chart showing the effects of the multiplex amplification method provided in this embodiment of the invention with traditional multiplex PCR. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] Multiplex PCR amplification is a commonly used technique for rapidly obtaining multiple target DNA fragments. However, when a large number of primers are introduced into a single reaction system, significant amplification heterogeneity and nonspecificity are often caused by primer interference and differences in amplification efficiency. Extensive experimental optimization of primer combinations and reaction conditions is required, severely hindering its clinical application and promotion. To address this, this invention innovatively proposes a novel strategy combining specific multi-target molecular markers with universal primers for amplification. By separating the primer recognition and amplification processes, it significantly reduces bias and nonspecificity caused by primer interactions, improves amplification uniformity and sensitivity, and establishes a simple, efficient, widely applicable, and clinically translational targeted multi-fragment equivalent amplification technique and gene detection method. The effectiveness and practicality of the technology have been verified through applications in genetic disease detection and pathogen detection. A supporting bioinformatics analysis algorithm has been developed to systematically evaluate its application value in scientific research and clinical practice.

[0030] Specifically, such as Figure 1 As shown, in one embodiment of the present invention, a multiplex amplification method is provided, which is a three-step multiplex amplification method, specifically including the following steps:

[0031] S1. Upstream primer labeling reaction: A mixture of upstream primers designed for multiple target regions is used to linearly amplify the target template and purify it to obtain the first product; the 5' end of the upstream primer contains a universal primer sequence and the 3' end contains a specific target recognition sequence.

[0032] S2. Downstream primer labeling reaction: The first product is linearly amplified using a mixture of downstream primers designed for multiple target regions, and the second product is purified; the 5' end of the downstream primer contains a universal primer sequence, and the 3' end contains a specific target recognition sequence.

[0033] S3. Universal primer homogenization amplification: Using universal primers designed based on the universal primer sequence, the second product is uniformly amplified exponentially.

[0034] In practical applications, the target template is generally template DNA. The process involves using a mixture of upstream primers designed for multiple target regions to linearly amplify the target template and purify it to obtain the first product. Step S1 specifically includes:

[0035] In a standard PCR reaction system (including template DNA, DNA polymerase, dNTPs, and reaction buffer), a forward primer mix (FPM) designed for multiple target regions is added. The forward primer contains a universal primer sequence at its 5' end and a specific target recognition sequence at its 3' end; the forward primer consists of both a universal primer sequence and a specific target recognition sequence. The PCR reaction system containing the forward primer mix is ​​then placed in a thermal cycler for 1-5 cycles of linear amplification (pre-denaturation 98℃ 30 s; cycles 1-5: 98℃ 10 s, 60℃ 10 s, 72℃ 30 s / kb; final extension 72℃ 3 min; storage 4℃). During this process, the forward primer binds to the template DNA, and after extension by DNA polymerase, the universal primer sequence is labeled upstream of the target region. By controlling the number of cycles, amplification bias caused by differences in primer binding efficiency can be effectively reduced. After the reaction was completed, the unreacted upstream primer mixture was removed by magnetic bead purification to obtain the first product.

[0036] Furthermore, using a mixture of downstream primers designed for multiple target regions, the first product is linearly amplified and purified to obtain the second product; the step S2, in which the 5' end of the downstream primers contains a universal primer sequence and the 3' end contains a specific target recognition sequence, specifically includes:

[0037] In the PCR reaction system containing the first product obtained in step S1, a reverse primer mix (RPM) designed for multiple target regions is added. The reverse primers contain a universal primer sequence at the 5' end and a specific target recognition sequence at the 3' end; each reverse primer consists of a universal primer sequence and a specific target recognition sequence. Then, using the same amplification conditions as in step S1, the PCR reaction system containing the reverse primer mix is ​​placed in a thermal cycler for 1-5 cycles of linear amplification (pre-denaturation 98℃ 30 s; cycles 1-5: 98℃ 10 s, 60℃ 10 s, 72℃ 30 s / kb; final extension 72℃ 3 min; storage 4℃). During this process, the reverse primers bind to and extend the template DNA, labeling the downstream of the target region with the universal primer sequence; simultaneously, the extension product covers the upstream primer region, thus ensuring that both ends of the target region are labeled with the universal primer sequence. Similarly, by controlling the number of cycles, amplification heterogeneity can be further reduced. After the reaction was completed, the unconsumed downstream primer mixture was removed by magnetic bead purification to obtain the second product.

[0038] In step S3, a universal primer is added to the second product marked with the paired-end universal primer sequence to perform uniform exponential PCR amplification. At this point, all target sequences have the same primer binding site, and the amplification efficiency is mainly affected by the initial template concentration and amplicon length, rather than depending on the differences in primer sequences for multiple targets. This achieves truly simultaneous and uniform amplification of multiple targets. This step can be performed for 15-35 cycles (pre-denaturation 98℃ 30 s; 15-35 cycles: 98℃ 10 s, 60℃ 10 s, 72℃ 30 s / kb; final extension 72℃ 5 min; storage at 4℃).

[0039] In a preferred embodiment of the present invention, the design of universal primer sequences should meet the following conditions:

[0040] It has no specific binding site to the target template to avoid non-specific amplification;

[0041] By optimizing primer design to reduce secondary structures and primer dimer formation, the robustness and reproducibility of the amplification reaction can be ensured.

[0042] It has a high Tm value (≥60℃) to reduce primer mismatch and nonspecific reactions.

[0043] In a preferred embodiment of the present invention, to achieve high coverage and high integrity of different types of genomic targets, the present invention further proposes a differentiated amplification strategy based on the above-mentioned universal primer amplification system, which may specifically include:

[0044] (1) Multiplex amplification of discrete multi-target regions: For multi-target regions that are discretely distributed and short in length in the genome, conventional multiplex amplification strategies can be used. By using multiple sets of specific primers, the above three-step multiplex amplification method can be used to achieve parallel amplification of multiple discontinuous target regions, so as to achieve the purpose of simultaneously detecting multiple genes or sites.

[0045] (2) Overlapping amplification (shingled amplification) strategy for continuous long fragments: For the detection needs of long fragments of single genes or continuous genomic regions, design overlapping amplicon primer sets (such as... Figure 1 As shown in the figure, multiple overlapping PCR (Tiling PCR) is performed. This strategy achieves unbiased and complete coverage of target regions ranging from tens to hundreds of kb in length by designing multiple sets of short amplicons (each amplicon is hundreds to thousands of bp in length) in tandem.

[0046] The resulting amplification products can be adapted for sequencing on second-generation short-read sequencing platforms or third-generation long-read sequencing platforms. For short-read sequencing results, a de novo assembly strategy can be used to reconstruct each short amplicon sequence into a continuous sequence of the target region, thereby achieving high-precision reconstruction at the biallelic level and detection of complex structural variations (such as insertions, deletions, and rearrangements).

[0047] Through the flexible combination of the above amplification strategies, the multiplex amplification system provided in this embodiment of the invention can be applied to the detection of multiple target genes / pathogens and the deep analysis of long fragments of single genes, which significantly improves the versatility, coverage and accuracy of multiplex amplification technology in the field of complex gene detection.

[0048] In another embodiment of the present invention, a multiplex amplification system is also provided for implementing the above-described multiplex amplification method, comprising DNA polymerase, dNTPs, and reaction buffer, and further comprising one or more upstream and downstream primers; wherein the 5' end of the upstream primer contains a universal primer sequence and the 3' end contains a specific target recognition sequence; the 5' end of the downstream primer contains a universal primer sequence and the 3' end contains a specific target recognition sequence. It should be noted that the universal primer sequences contained in the upstream and downstream primers designed for the same target region can be the same, and the universal primer sequences contained in the upstream or downstream primers designed for different target regions in the same multiplex amplification system are all the same. The specific universal primer sequences used in practical applications are shown in SEQ ID NO:1 of the sequence listing; furthermore, the specific target recognition sequences contained in the upstream and downstream primers designed for the same or different target regions are different. It should be noted that the embodiments of the present invention only exemplarily list some pathogenic genes and primer sequences used for pathogen detection, but are not limited thereto. Those skilled in the art can design universal primer sequences and specific target recognition sequences according to the actual detection target.

[0049] like Figure 2 As shown, in practical applications, the multiplex amplification method using specific multi-target molecular markers combined with universal primers employed in the embodiments of this invention can be widely applied to various clinical testing scenarios such as molecular diagnosis of genetic diseases and rapid detection of pathogens. The following embodiments verify the feasibility, stability, and versatility of the system provided in the embodiments of this invention in different application fields.

[0050] Example 1: Detection of Unknown Genetic Disease Mutations – Precise Diagnosis of STK11 Gene Whole Exon:

[0051] For the STK11 gene associated with Peutz-Jeghers Syndrome (PJS), multiple overlapping specific amplification primer pairs were designed (the upstream primer contains the universal primer sequence shown in SEQ ID NO:1, specifically any one of SEQ ID NO:2-9; the downstream primer contains the universal primer sequence shown in SEQ ID NO:1, specifically any one of SEQ ID NO:10-17; wherein the upstream primer sequences shown in SEQ ID NO:2-9 are paired with the downstream primer sequences shown in SEQ ID NO:10-17 respectively), achieving seamless coverage of the full-length gene (22kb). Using the three-step multiplex amplification method provided in this embodiment (i.e., upstream primer labeling reaction, downstream primer labeling reaction, and universal primer homogenization amplification), amplification products with universal primer sequences at both ends were obtained.

[0052] The amplified products obtained can be directly used for downstream detection and analysis on the Illumina second-generation sequencing platform and the Oxford Nanopore Technologies (ONT) third-generation sequencing platform. Sequencing results show that high-quality data with an average sequencing depth ≥500× can be obtained on both platforms, indicating that the system has good compatibility and amplification consistency with different sequencing platforms.

[0053] To verify the performance of the method provided in this embodiment of the invention, the same amplification primers were selected for the following tests: (1) amplification using the three-step multiplex amplification method of this embodiment of the invention; and (2) amplification using the traditional one-step multiplex PCR method. The primers used in the traditional multiplex PCR method contained only specific target recognition sequences and did not contain universal primer sequences. The amplification products were sequenced using the ONT platform with the same number of reads, and the results are as follows: Figure 3 As shown, the results indicate that the multiplex amplification method provided in this embodiment of the invention can significantly improve amplification uniformity and coverage, while significantly reducing the proportion of nonspecific amplification products.

[0054] The validation experiment selected a number of clinically diagnosed PJS patients (5-10 cases) carrying the known STK11 pathogenic mutation gene as the positive validation group. The test results showed that the mutation sites detected in this embodiment of the invention were completely consistent with the results of whole exome sequencing (WES), indicating that the multiplex amplification method provided in this embodiment of the invention has high accuracy and high specificity.

[0055] Furthermore, the multiplex amplification method provided in this invention was successfully applied to several suspected PJS cases that were WES negative, successfully detecting novel mutation sites not detected by WES. This result demonstrates that the multiplex amplification method provided in this invention has significant advantages in identifying cryptic mutations, complex structural variations, and difficult-to-amplify regions, showcasing its application potential in the field of precision diagnosis of clinical genetic diseases.

[0056] Example 2: Multi-target high-throughput screening of known genetic disease mutation sites—application of neonatal hearing impairment gene panel:

[0057] A multiplex detection panel covering common deafness-causing gene mutations was constructed, containing 26 high-frequency mutation sites, including genes such as GJB2, SLC26A4, and mitochondrial rRNA. The design of each amplification target takes into account different mutation types (point mutations, small fragment insertions / deletions) to comprehensively evaluate the system's detection performance.

[0058] The multiplex amplification method provided in this embodiment of the invention is compared with the traditional multiplex PCR method. Both groups used the same template and primer sequences (specific target recognition sequences) and amplified under the same reaction conditions. The primers used in the traditional multiplex PCR method only contain specific target recognition sequences and do not contain universal primer sequences. In contrast, the primers used in the multiplex amplification method provided in this embodiment of the invention not only contain specific target recognition sequences (consistent with those used in the traditional multiplex PCR method) but also contain the universal primer sequence shown in SEQ ID NO:1. The results show that the amplification products of this embodiment of the invention have higher coverage uniformity, significantly reduced non-specific amplification, and better target detection accuracy and repeatability than the traditional method. These results demonstrate that the multiplex amplification method provided in this embodiment of the invention can maintain high sensitivity and high consistency in parallel detection of multiple mutation sites and is suitable for large-scale gene panel detection.

[0059] Example 3: Rapid multiplex detection of pathogens using RPA isothermal amplification combined with real-time sequencing:

[0060] To verify the application capability of the multiplex amplification method provided in this invention in the field of rapid pathogen detection, two multipathogen detection panels were constructed:

[0061] 1. Respiratory Pathogen Panel: Covers 10-12 common respiratory pathogens, including influenza A / B virus, SARS-CoV-2, RSV, coronavirus, Mycoplasma pneumoniae, and Chlamydia pneumoniae; wherein, the respiratory pathogen detection primers used in the multiplex amplification method provided in this embodiment of the invention not only contain specific target recognition sequences (which can be consistent with the detection primer sequences used in traditional multiplex PCR methods), but also contain the universal primer sequences shown in SEQ ID NO:1 of the sequence listing;

[0062] 2. Gastrointestinal Pathogen Panel: Covers 8 major viruses including rotavirus, norovirus (GI / GII), adenovirus, astrovirus, and zaruzin virus; wherein, the gastrointestinal pathogen detection primers used in the multiplex amplification method provided in this embodiment of the invention not only contain specific target recognition sequences (which can be consistent with the detection primer sequences used in traditional multiplex PCR methods), but also contain the universal primer sequences shown in SEQ ID NO:1 of the sequence listing.

[0063] After collecting nasopharyngeal swabs or fecal samples, RNA-DNA co-extraction and reverse transcription are performed. The resulting cDNA / DNA samples are then enriched for specific targets using the multiplex amplification method provided in this invention. Isothermal amplification techniques, such as the RPA (Recombinase Polymerase Amplification) isothermal amplification system, are used during the amplification process, replacing thermal cycling amplification. Rapid amplification is achieved at a constant temperature of 39°C for 10-20 minutes. The reaction system requires only simple heating equipment, making it suitable for grassroots or on-site testing.

[0064] After rapid library construction (ONT Rapid Adapter method), RPA products are subjected to real-time sequencing using a portable MinION sequencer. Targeted real-time analysis algorithms are employed for simultaneous sequencing and analysis, enabling real-time base identification and pathogen comparison, allowing sample detection to be completed within hours. The combination of the multiplex amplification method and real-time sequencing provided in this invention enables rapid and sensitive detection of multiple pathogens, significantly shortening the detection cycle and demonstrating good scalability and application prospects.

[0065] The above embodiments demonstrate that the multiplex amplification method provided by this invention exhibits good amplification uniformity, specificity, detection accuracy, and versatility in different detection scenarios. This method is applicable simultaneously to complex genetic disease mutation detection, multi-gene screening, and rapid on-site diagnosis of multiple pathogens, possessing significant technical advantages and industrialization potential.

[0066] In summary, compared with existing traditional multiplex PCR methods, the multiplex amplification method provided in this invention has the following significant technical advantages and beneficial effects:

[0067] 1. Significantly Improved Amplification Uniformity: This invention, through the design of specific multi-target molecular markers and universal primers for uniform amplification, effectively eliminates the competition effect between different primers, making the amplification efficiency of each target more consistent and significantly reducing the deviation in amplification depth. This feature reduces resource waste caused by local over-amplification during sequencing, improves sequencing economy, and ensures that all target regions are adequately covered, thereby avoiding missed detections due to uneven amplification.

[0068] 2. Significantly reduced nonspecific amplification: By introducing optimized universal primer sequences and a short-range linear amplification strategy, the probability of mismatch amplification and primer dimer formation was significantly reduced, improving the specificity and stability of amplification. Due to the reduction in primer dimers, sequencing resources can be concentrated on the target fragment, improving data utilization and analytical accuracy.

[0069] 3. Strong compatibility with sequencing platforms: The amplified products obtained by this invention are directly compatible with mainstream high-throughput sequencing platforms, including next-generation sequencing (Illumina) and third-generation sequencing (Oxford Nanopore). Library construction and sequencing can be completed without additional adaptation or modification. Combining shingled overlap amplification and de novo assembly strategies, next-generation sequencing can also achieve accurate identification of complex structural variations, expanding the detection capabilities of traditional short-read platforms.

[0070] 4. Superior Coverage: By designing overlapping target amplicons, continuous and seamless coverage of ultra-long gene regions is achieved, ensuring the integrity of the target sequence. This invention is suitable for detecting complex structural variations such as large fragment deletions, insertions, and gene rearrangements, and is particularly suitable for amplification detection of structurally complex regions or regions with extreme GC content.

[0071] 5. Wide range of applications: The multiplex amplification system of this invention has high versatility and scalability, and can be widely used in many fields such as genetic disease gene detection, multi-gene joint screening, multi-pathogen detection, molecular typing analysis and targeted sequencing library construction, and has good prospects for scientific research and clinical promotion.

[0072] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A multiplex amplification method for purposes other than disease diagnosis and treatment, characterized in that, Includes the following steps: Upstream primer labeling reaction: A mixture of upstream primers designed for multiple target regions is used to linearly amplify the target template. After the reaction, the mixture is purified to remove unreacted upstream primers to obtain the first product. The 5' end of the upstream primers contains a universal primer sequence, and the 3' end contains a specific target recognition sequence. Downstream primer labeling reaction: The first product is linearly amplified using a mixture of downstream primers designed for multiple target regions. After the reaction, the product is purified to remove unreacted downstream primers to obtain the second product. The 5' end of the downstream primers contains a universal primer sequence, and the 3' end contains a specific target recognition sequence. Universal primer homogenization amplification: The second product is uniformly amplified exponentially using universal primers designed based on the universal primer sequence; the universal primer sequence is the same as the universal primer sequence contained at the 5' end of the upstream primer and the universal primer sequence contained at the 5' end of the downstream primer; The universal primer sequence has no specific binding site to the target template, and the primer design is optimized to reduce secondary structure and primer dimer formation.

2. The multiplex amplification method according to claim 1, characterized in that, The universal primer sequence is shown in the sequence listing SEQ ID NO:

1.

3. The multiplex amplification method according to claim 1, characterized in that, The steps of linearly amplifying the target template and purifying it to obtain the first product using a mixture of upstream primers designed for multiple target regions specifically include: In a PCR reaction system containing the target template, a mixture of upstream primers designed for multiple target regions is added, and a linear amplification reaction is performed for 1-5 cycles. Universal primer sequences are labeled upstream of the target region. After the reaction, the unreacted upstream primer mixture is removed by purification with magnetic beads to obtain the first product.

4. The multiplex amplification method according to claim 1, characterized in that, The steps of linearly amplifying the first product and purifying it to obtain the second product using a mixture of downstream primers designed for multiple target regions specifically include: In a PCR reaction system including the first product, a mixture of downstream primers designed for multiple target regions is added, and a linear amplification reaction is performed for 1-5 cycles. Universal primer sequences are labeled downstream of the target regions, and the extension products cover the upstream primer regions, so that both ends of the target regions are labeled with universal primer sequences. After the reaction is completed, the unreacted downstream primer mixture is removed by purification with magnetic beads to obtain the second product.

5. The multiplex amplification method according to claim 1, 2, 3, or 4, characterized in that, It also includes the following steps: By combining shingled amplification, amplicon sequencing, and de novo assembly strategies, complete coverage of long target regions can be achieved, and complex structural variations can be detected through short-read sequencing.

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