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CN-121975965-A - Targeted sequencing primer composition, kit and library construction method for simultaneously detecting 41 infectious disease pathogens

CN121975965ACN 121975965 ACN121975965 ACN 121975965ACN-121975965-A

Abstract

The invention relates to a targeted sequencing primer composition for simultaneously detecting 41 infectious disease pathogens, a kit and a library building method. The primer composition comprises a forward specific primer group, a reverse specific primer group, a bridge primer, a universal primer 1 and a universal primer 2, and is specifically shown as SEQ ID NO. 1-139. The invention also provides a kit containing the primer composition and a method for constructing an amplicon sequencing library by using single-tube three primers for non-diagnostic purposes. The invention designs 68 pairs of specific primers for covering viruses, bacteria and parasites aiming at 41 infectious disease pathogens legal in China, and realizes comprehensive and specific pathogen coverage. And through bioinformatics verification and experimental verification, the high specificity of the primer is ensured, the target gene conservation region can be accurately identified, and non-specific amplification is effectively avoided.

Inventors

  • ZHANG ZHONGFA
  • Cui Chaiyan
  • LI QIANG

Assignees

  • 山东省公共卫生临床中心
  • 山东省大健康精准医疗产业技术研究院

Dates

Publication Date
20260505
Application Date
20260407

Claims (10)

  1. 1. A targeted sequencing primer composition for simultaneous detection of 41 infectious disease pathogens, comprising a forward specific primer set, a reverse specific primer set, a bridge primer, a universal primer 1, and a universal primer 2: the forward specific primer group comprises sequences shown as SEQ ID NO. 1-68; The reverse specificity primer group comprises sequences shown as SEQ ID NO. 69-136; The sequence of the bridge primer is shown as SEQ ID NO. 137; the sequence of the universal primer 1 is shown as SEQ ID NO. 138; The sequence of the universal primer 2 is shown as SEQ ID NO. 139.
  2. 2. The primer composition of claim 1, wherein each sequence in the forward specific primer set is formed by ligating a first sequencing tag sequence, an upstream sequence of a sample tag sequence barcode, and an amplicon specific forward primer in sequence from a 5 'end to a 3' end; In the reverse specificity primer group, each sequence is formed by sequentially connecting a second sequencing tag sequence, a downstream sequence of a sample tag sequence barcode and an amplicon specificity reverse primer from a 5 'end to a 3' end; The bridge primer sequentially comprises a second sequencing joint sequence and a second sequencing tag sequence from the 5 'end to the 3' end; the universal primer 1 sequentially comprises a first sequencing joint sequence and a first sequencing tag sequence from a 5 'end to a 3' end; the universal primer 2 is a second sequencing adapter sequence.
  3. 3. The primer composition according to claim 2, wherein, the method is characterized in that the first sequencing tag sequence is as follows: 5'-TCGTCGGCAGCGTC-3'; the second sequencing tag sequence is 5'-GTCTCGTGGGCTCGG-3'; The first sequencing linker sequence is 5'-AATGATACGGCGACCACCGAGATCTACAC-3'; The second sequencing linker sequence is 5'-CAAGCAGAAGACGGCATACGAGAT-3'; the upstream and downstream sequences of the sample tag sequence barcode are used to distinguish between amplified products of different origins after sequencing.
  4. 4. The primer composition of claim 1, wherein the primer in the primer composition corresponds to 41 legal infectious diseases, infectious disease pathogens as shown in the following table: in the primer composition, the molar concentration ratio of the forward specific primer group to the reverse specific primer group to the bridge primer is 2:1:2.
  5. 5. A kit for simultaneous detection of 41 infectious agents, comprising the primer composition of claim 1.
  6. 6. The kit of claim 1, further comprising nucleic acid extraction reagents, nucleic acid amplification reagents, purification reagents, buffers, and nuclease-free water.
  7. 7. Use of the primer composition of claim 1 or the kit of claim 6 for the preparation of 41 legal infectious disease pathogen detection products.
  8. 8. A method for constructing an amplicon sequencing library with a single tube three primer for non-diagnostic purposes, comprising the steps of: (1) Extracting total nucleic acid in a sample to be detected, and reversely transcribing RNA into cDNA; (2) Performing a first round of multiplex PCR amplification by using the cDNA obtained in the step (1) as a template and using a forward specific primer group, a reverse specific primer group and a bridge primer, and purifying to obtain a first round of amplification product; (3) And (3) taking the purified first round of amplification product as a template, performing second round of PCR amplification by using the universal primer 1 and the universal primer 2, and purifying to obtain an amplicon sequencing library which can be used for subsequent high-throughput sequencing.
  9. 9. The method according to claim 8, wherein in the step (2), the reaction system of the first round multiplex PCR amplification is that the template nucleic acid (cDNA) is 50 ng, the forward specific primer set (1. Mu.M) is 2. Mu.L, the reverse specific primer set (1. Mu.M) is 1. Mu.L, the bridge primer set (1. Mu.M) is 2. Mu.L, platinumTM Multiplex PCR MASTER Mix is 25. Mu.L, and the nuclease-free water is added to the total volume of 50. Mu.L; The first round multiplex PCR amplification was performed with a pre-denaturation at 95℃of 3 min followed by 25 cycles each consisting of denaturation at 95℃of 30 s followed by annealing/extension at 61℃59℃and 57℃of 2 min in the same cycle and a final extension at 72℃of 10 min.
  10. 10. The method according to claim 8, wherein in the step (3), the reaction system of the second round of PCR amplification is that the first round of amplification product is 5. Mu.L, the universal primer 1 (10. Mu.M) is 5. Mu.L, the universal primer 2 (10. Mu.M) is 5. Mu.L, 2X KAPA HiFi HotStart ReadyMix. Mu.L, and nuclease-free water is added to a total volume of 50. Mu.L; The procedure for the second round of PCR amplification was 95℃pre-denaturation 3 min followed by 8 cycles, each cycle consisting of 95℃denaturation 30s, 60℃annealing 30s, 72℃extension 30s, and finally 72℃final extension 5 min.

Description

Targeted sequencing primer composition, kit and library construction method for simultaneously detecting 41 infectious disease pathogens Technical Field The invention belongs to the technical field of molecular biology detection, and particularly relates to a targeted sequencing primer composition, a kit and a library construction method for simultaneously detecting 41 infectious disease pathogens, and particularly relates to a targeted sequencing primer composition, a kit and a library construction method which are based on targeted high-throughput sequencing and can simultaneously detect 41 infectious disease pathogens. Background Infectious diseases form a continuous threat to global public health, and rapid, accurate and comprehensive pathogen identification is a key to effective prevention, control and treatment. Traditional pathogen detection methods, such as morphological observation, culture methods, immunological detection and single PCR, have the limitations of low sensitivity, long time consumption, limited flux or single target detection at a time, and are difficult to deal with the screening requirements of mixed infection or new pathogens. The metagenome second generation sequencing (mNGS) technology does not need to be preset, can detect all microbial nucleic acids in a sample without bias, but has the problems of large host background interference, high cost, complex biological information analysis and the like, and limits the conventional clinical application of the metagenome second generation sequencing (mNGS). The targeted Next-Generation Sequencing, tNGS technology realizes that sequencing data volume and analysis difficulty are remarkably reduced while high sensitivity and specificity are maintained by performing primer design and high-throughput sequencing on a specific pathogen target region, and becomes a powerful tool for diagnosing infectious diseases. However, the existing tNGS technical scheme still has two major challenges, namely 1) complex primer set design for large-scale pathogens (such as covering all legal infectious diseases), and uneven amplification efficiency caused by primer-primer interaction, and 2) complicated conventional two-step amplicon library-building process, which requires independent purification steps for each sample, and when large-scale samples are processed, the operation is complex, the reagent consumption is large, and cross contamination is easy to introduce. Therefore, there is a need to develop a high-efficiency tNGS detection scheme that can cover important infectious disease pathogens at one time, has a simplified library-building process, and is suitable for batch detection. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a targeted sequencing primer group, a kit and a library construction method for simultaneously detecting 41 infectious disease pathogens. The primer composition can detect 41 legal infectious disease pathogens at one time, greatly simplifies the operation flow, does not need to design a primer and a database for each sample independently, and greatly saves time and labor cost. In order to achieve the above purpose, the invention adopts the following technical scheme: In a first aspect, the present invention provides a targeted sequencing primer composition for simultaneous detection of 41 infectious disease pathogens, comprising a forward specific primer set, a reverse specific primer set, a bridge primer, a universal primer 1 and a universal primer 2: the forward specific primer group comprises sequences shown as SEQ ID NO. 1-68; The reverse specificity primer group comprises sequences shown as SEQ ID NO. 69-136; The sequence of the bridge primer is shown as SEQ ID NO. 137; the sequence of the universal primer 1 is shown as SEQ ID NO. 138; The sequence of the universal primer 2 is shown as SEQ ID NO. 139. According to the invention, in the forward specific primer group, each sequence is formed by sequentially connecting a first sequencing tag sequence, an upstream sequence of a sample tag sequence barcode and an amplicon specific forward primer from a 5 'end to a 3' end; In the reverse specificity primer group, each sequence is formed by sequentially connecting a second sequencing tag sequence, a downstream sequence of a sample tag sequence barcode and an amplicon specificity reverse primer from a 5 'end to a 3' end; The bridge primer sequentially comprises a second sequencing joint sequence and a second sequencing tag sequence from the 5 'end to the 3' end; the universal primer 1 sequentially comprises a first sequencing joint sequence and a first sequencing tag sequence from a 5 'end to a 3' end; the universal primer 2 is a second sequencing adapter sequence. Further preferably, the first sequencing tag sequence is 5'-TCGTCGGCAGCGTC-3'; the second sequencing tag sequence is 5'-GTCTCGTGGGCTCGG-3'; The first sequencing linker sequence is 5'-AATGATACGGCGACCACCGAGATCTACAC-3'; The second s