Nested PCR method and use thereof

Abstract

A nested PCR method and the use thereof, and an improved targeted detection and library building method formulated on the basis of a nested PCR library building method. The method uses the nucleic acid cleavage activity of a USER enzyme, combined with a Taq DNA polymerase or an enzyme having both a DNA polymerization activity and a 5'-3' exonuclease activity, to establish a method for primer removal, to replace a purification step between a first round of PCR and a second round of PCR, thereby successfully solving the problem of cross contamination among samples caused by the purification operation of nested PCR. In addition, the method can effectively remove non-specific amplification products, improves the specificity of targeted amplification, and has a simpler experimental process. The nested PCR method can be applied to a targeted library building platform and a sequencing platform.

Description

A nested PCR method and its application Technical Field

[0001] The present disclosure relates to the field of biomolecules, and in particular, to a nested PCR method and application. Background Art

[0002] Infectious diseases are numerous and complex, and are a leading cause of death. Early detection, diagnosis, and treatment are key to preventing and treating infectious diseases. Traditional pathogen detection methods, including culture and immunoassays, suffer from numerous issues, including low sensitivity, long detection cycles, and a limited pathogen spectrum. Molecular detection technologies based on high-throughput sequencing have developed rapidly in this field in recent years, offering a broader pathogen detection spectrum, higher sensitivity, and greater throughput than traditional detection methods.

[0003] Currently, pathogen detection methods based on high-throughput sequencing mainly include metagenomic detection (mNGS) and targeted pathogen detection (target NGS, tNGS). Metagenomic detection is the one-time detection of all microorganisms in a sample. This method is of great significance for the diagnosis of patients with difficult and critical clinical infections because it can detect unknown pathogens. However, for other clinical infection detection, there are problems such as long detection cycle, high cost, and low sensitivity in detecting low-abundance pathogens. Targeted pathogen detection (tNGS) combines the advantages of multiplex PCR amplification and high-throughput sequencing to detect known pathogens and their virulence / resistance genes. With its advantages of low cost and high detection sensitivity, it is rapidly seizing the pathogen detection market.

[0004] Nanopore sequencing technology is more suitable for rapid identification of pathogens of clinical infectious diseases than second-generation sequencing due to its long length, fast sequencing speed, and real-time analysis. Targeted pathogen detection based on the nanopore sequencing platform first requires the construction of a targeted library. Currently, there are two main library construction methods:

[0005] Method 1: One-step PCR ligation library construction

[0006] First, a first round of PCR amplification of the sample nucleic acid is performed using specific primers. The amplified products are then purified. The purified amplified products are then end-repaired and subjected to a second round of nucleic acid purification. Next, sample tags are ligated to each sample, and the ligated products are purified a third time. Finally, the purified ligated products are pooled and adapter-ligated to obtain the final sequenceable library.

[0007] This method has the following problems: (1) When multiple targets need to be amplified by PCR at the same time, a large number of primers exist in the same system. Only using specific primers for one PCR will easily produce more non-specific amplification products, including abnormal hybrids formed between primers and between primers and templates, thereby affecting the amplification and detection of the target; (2) After the first round of PCR amplification is completed and after the end repair, nucleic acid purification is required. At this time, the sample has not yet been added with a sample label. When multiple sample libraries are constructed, the aerosols generated during the PCR amplification process will be mixed into each sample during the purification operation, which can easily lead to cross-contamination between samples and affect the accuracy of pathogen detection; (3) The entire experimental process takes 4 hours and requires 3 rounds of nucleic acid purification. The library construction time is long and the operation is relatively cumbersome.

[0008] Method 2: Two-step PCR library construction

[0009] First, the method adds a universal sequence to the specific primers used in the first round of PCR. The forward primer contains the sequence US-a: 5'-TTTCTGTTGGTGCTGATATTGC-3', and the forward primer contains the sequence US-b: 5'-ACTTGCCTGTCGCTCTATCTTC-3'. This universal sequence complements the barcode primers used in the second round of PCR. Sample nucleic acid is amplified in the first round of PCR using specific primers carrying the universal sequence, and the amplified product is purified to remove unreacted primers. The purified first-round amplified product is then amplified in the second round of PCR using the barcode primers, which are also purified. The barcode primers can consist of 96 different barcode sequences and the reverse complement of the universal sequence. Finally, the purified second-round PCR products are pooled and adapter-ligated to generate the final sequence-ready library.

[0010] This method uses a two-step PCR amplification method. First, a relatively small number of cycles are used to perform a first round of PCR amplification on the sample nucleic acid to obtain a small amount of specific amplification products. The second round of PCR amplification uses the specific products of the first round of PCR as a template for a second round of amplification. Compared with the one-step PCR method, this amplification method can significantly improve the specificity of the amplification products. However, the following problems still exist: (1) After completing the first round of PCR amplification, the sample needs to be purified to remove the primers that have not reacted completely. At this time, the sample has not yet introduced the sample label. Although the number of cycles in the first round of PCR in this method is relatively small, the aerosol contamination problem is relatively less serious. However, during the purification operation, even a small amount of aerosol can easily mix into each sample, resulting in cross-contamination between samples. (2) The experimental process takes about 3 hours, which is shorter than the library construction time of method 1. However, because two steps of purification are still required, the operation is still relatively cumbersome when building a library for multiple samples.

[0011] Currently, there are two methods for targeted library construction suitable for nanopore sequencing platforms. The one-step PCR ligation library construction method is prone to produce a large number of nonspecific amplification products (Nonspecific products), and the entire process requires three rounds of nucleic acid purification (Purification). It is not only cumbersome to operate, but also very easy to introduce cross-contamination between samples during the purification operation; the two-step PCR library construction method can enhance the specificity of the amplification product and introduce sample tags while amplifying the pathogen target in the second step of PCR. It is more convenient and faster, with a shorter experimental process. It is the current mainstream targeted library construction method. However, this method still requires two rounds of nucleic acid purification and there is also the risk of introducing cross-contamination between samples during the purification process. For pathogen targeted detection, cross-contamination between samples is an important factor affecting the accuracy of the test results, which can easily lead to false positives in sample detection.

[0012] Cross-contamination between samples occurs in many PCR scenarios, especially when multi-step amplification of samples is required. Therefore, there is an urgent need for a PCR method that can eliminate cross-contamination between samples and improve the specificity of target sequence amplification.

[0013] Summary of the Invention

[0014] In order to solve at least one of the above technical problems, the present disclosure provides a nested PCR method in a first aspect, comprising:

[0015] (a) performing a first round of PCR on the sample to be tested using a first primer with a single-base restriction site to obtain a first round of PCR product;

[0016] (b) adding enzyme 1, enzyme 2, and a tag primer to the first-round PCR product to perform a second-round PCR reaction;

[0017] The enzyme 1 is used to cut the sequence with a single-base restriction site, and the enzyme 2 is used to repair the single-base gap formed by the enzyme 1 in the first-round PCR product.

[0018] In response to the problems of existing multi-step PCR methods: the easy generation of a large number of non-specific amplification products, thereby affecting the amplification and detection of the target; the purification operation during the experiment easily leads to cross-contamination between samples, affecting the detection accuracy of the target; there are many purification steps and the operation is relatively cumbersome. The present disclosure provides an improved nested PCR method that can eliminate the cross-contamination problem between samples, while further improving the amplification specificity of the target and simplifying the experimental process. It is an efficient, convenient, and cross-contamination-avoiding nested PCR method.

[0019] According to a specific embodiment of the present disclosure, the sequence with the single-base restriction site includes the first primer pair and the first-round PCR product.

[0020] According to a specific embodiment of the present disclosure, any one primer in the first primer pair includes a target-specific sequence and a universal sequence, wherein the target-specific sequence is complementary to a portion of the nucleic acid sequence of the sample to be tested, and the universal sequence is complementary to a portion of the sequence of the tag primer. The 5'-3' sequence of the first primer pair includes the universal sequence + the target-specific sequence.

[0021] According to specific embodiments of the present disclosure, the tag primer includes a tag sequence and a sequence complementary to a universal sequence. The "sequence complementary to the universal sequence" in the tag primer refers to the "partial sequence of the tag primer" within the sequence complementary to the universal sequence. According to specific embodiments of the present disclosure, the target-specific sequence contains the single-base restriction site.

[0022] According to a specific embodiment of the present disclosure, the universal primer contains the single-base enzyme cleavage site.

[0023] According to a specific embodiment of the present disclosure, both the targeting specific sequence and the universal sequence contain the single-base enzyme cleavage site.

[0024] According to a specific embodiment of the present disclosure, the targeting specific sequence and the universal sequence each contain at least one single-base enzyme cleavage site.

[0025] According to a specific embodiment of the present disclosure, the sequence length of any one primer in the first primer pair is 36-48 nt.

[0026] According to a specific embodiment of the present disclosure, the targeting-specific sequence is 18-24 nt in length.

[0027] According to a specific embodiment of the present disclosure, the universal sequence is 18-24 nt in length.

[0028] According to a specific embodiment of the present disclosure, any one primer in the first primer pair contains at least one single-base restriction site.

[0029] According to a specific embodiment of the present disclosure, any one primer in the first primer pair contains 1-5 enzyme cutting sites.

[0030] According to a specific embodiment of the present disclosure, any one primer in the first primer pair contains two enzyme cutting sites.

[0031] According to a specific embodiment of the present disclosure, the single-base enzyme cleavage site includes modified ribonucleotides or deoxyribonucleotides.

[0032] According to a specific embodiment of the present disclosure, the single-base restriction site includes at least one of uracil deoxyribonucleotide, 5-hydroxyuracil deoxyribonucleotide, 5-hydroxymethyluracil deoxyribonucleotide, and 5-formyluracil deoxyribonucleotide.

[0033] According to a specific embodiment of the present disclosure, the enzyme 1 includes at least one of USER enzyme, UDG enzyme, hsMUG1 enzyme, and lytic enzyme Endo VIII.

[0034] According to a specific embodiment of the present disclosure, the enzyme 2 has both DNA polymerase activity and 5'-3' exonuclease activity.

[0035] According to a specific embodiment of the present disclosure, the enzyme 2 comprises Taq DNA polymerase or DNA polymerase I.

[0036] According to a specific embodiment of the present disclosure, in step (b), the second round of PCR reaction includes two incubation reactions and PCR reactions, wherein the first incubation reaction includes using enzyme 1 to cut the sequence with a single-base restriction site, and the second incubation reaction includes using enzyme 2 to repair the single-base gap formed in the first round of PCR product after being cut by enzyme 1.

[0037] According to a specific embodiment of the present disclosure, step (b) comprises the following steps:

[0038] Enzyme 1, enzyme 2, and tag primer were sequentially added to the first-round PCR product to carry out the second-round PCR reaction.

[0039] According to a specific embodiment of the present disclosure, step (b) comprises the following steps:

[0040] Enzyme 1, enzyme 2, and label primers are mixed and added to the first-round PCR product to perform a second-round PCR reaction.

[0041] According to a specific embodiment of the present disclosure, step (b) comprises the following steps:

[0042] After adding enzyme 1 and enzyme 2 to the first round PCR product for two incubation reactions, the tag primer is added to carry out PCR reaction.

[0043] According to a specific embodiment of the present disclosure, the temperature of the first incubation reaction is different from the temperature of the second incubation reaction. The present disclosure controls the occurrence of the two incubation reactions in the reaction system by controlling the temperatures of the two incubation reactions.

[0044] According to a specific embodiment of the present disclosure, the time of the first incubation reaction is not less than 5 minutes, and the temperature of the first incubation reaction is 20-45°C.

[0045] According to a specific embodiment of the present disclosure, the time of the second incubation reaction is 1-25 minutes, and the temperature of the second incubation reaction is 65°C-72°C.

[0046] The present disclosure provides an improved nested PCR method that can eliminate the problem of cross-contamination between samples, while further improving the amplification specificity of the target and simplifying the experimental process. It is an efficient, convenient, and cross-contamination-avoiding nested PCR method.

[0047] The second aspect of the present disclosure proposes the application of the above-mentioned nested PCR method in library construction.

[0048] A third aspect of the present disclosure provides a method for constructing a sequencing library, the method comprising:

[0049] (c) obtaining the second-round PCR product using the nested PCR method described above;

[0050] (d) Connecting the second-round PCR products to sequencing adapters to obtain a sequencing library.

[0051] The fourth aspect of the present disclosure proposes the application of the construction method of the third aspect in sequencing.

[0052] The fifth aspect of the present disclosure provides a library suitable for sequencing, which is prepared by the construction method of the third aspect.

[0053] According to a specific embodiment of the present disclosure, the library is suitable for sequencing by synthesis and nanopore sequencing methods.

[0054] A sixth aspect of the present disclosure provides a library construction kit, comprising:

[0055] Reagents configured to construct the library of the fifth aspect.

[0056] According to a specific embodiment of the present disclosure, the reagent includes at least one of a primer and an enzyme.

[0057] A seventh aspect of the present disclosure proposes the application of the above library construction kit in nanopore targeted detection.

[0058] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0060] FIG1 shows a flow chart of a method for building a targeted library for pathogen detection according to a specific embodiment of the present disclosure;

[0061] FIG2 shows the results of the primer digestion system condition optimization test in Example 1 of the present disclosure;

[0062] FIG3 shows the results of the Taq DNA polymerase screening experiment in Example 2 of the present disclosure;

[0063] FIG4 shows the results of the optimization test of the amount of Taq DNA polymerase added and the reaction time in Example 3 of the present disclosure.

[0064] Detailed Description of the Invention

[0065] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0066] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0067] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

[0068] In the present disclosure, the terms “comprise” or “include” are open expressions, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0069] In this disclosure, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0070] Definitions and Explanations of Terms

[0071] In the present disclosure, the term "USER enzyme" refers to an enzyme that can be used for nucleic acid cleavage. USER enzyme is a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII. UDG catalyzes the cleavage of uracil bases, forming an abasic site while keeping the phosphodiester backbone structure intact. The lyase activity of Endo VIII breaks the phosphodiester bonds at the 3' and 5' ends of the abasic site, releasing a base-free deoxyribose, i.e., USER enzyme can break nucleic acids by cleaving U bases.

[0072] In the present disclosure, the term "UDG enzyme" (Uracil DNA Glycosylase) can effectively catalyze the hydrolysis of the N-glycosidic bond between the uracil base and the sugar-phosphate backbone in single-stranded or double-stranded DNA.

[0073] In the present disclosure, the term "nested PCR" refers to a technique of performing two or more rounds of PCR amplification using two or more sets of PCR primers.

[0074] The problem to be solved by the present disclosure is to provide a new nested PCR method. Compared with the existing technology, it is necessary to focus on solving the problem of cross-contamination between samples during multiple rounds of PCR, and at the same time, it is necessary to ensure the amplification specificity of the nested PCR method for the target sequence and the simplicity of the process. In order to ensure the amplification specificity of the nested PCR method for the target sequence, the PCR method in the two-step PCR library construction method is preferably used. The main problem of this method is the contamination problem introduced by the purification step after the first round of PCR. To avoid contamination, the best way is to remove this purification step. The inventors tried to directly add the tag primers to the second round of PCR without purification of the first round PCR products, but found that this would result in the production of a large number of non-specific amplification products, mainly from the large number of primer dimers formed between the primers. This is mainly because the universal sequence contained in the specific primers of the first round of PCR can complement part of the sequence in the tag primers of the second round of PCR, thereby producing a large number of non-specific amplification products between the primers. To solve the above problems, the inventors hope to use a new method that can eliminate the residual primers in the first round of PCR without purification.

[0075] According to a specific embodiment of the present disclosure, the inventors incorporated U bases into all specific primers in the first round of PCR, that is, using U bases to replace some T bases in the primers. The sample nucleic acid was amplified in the first round of PCR using U-containing primers. Then, an enzyme that can specifically cleave nucleotides containing U bases was added to the amplified product for incubation. The enzyme that can specifically cleave nucleotides containing U bases includes but is not limited to USER enzyme, SMUG1 enzyme, or UDG enzyme. It should be noted that any enzyme known in the art that can achieve similar functions is suitable for the present disclosure. Here, the inventors chose USER enzyme for a short incubation. After cleavage by the USER enzyme, the primers in single-stranded form will be chopped up, and the double-stranded target amplification products (Target amplicons) will produce multiple single-base gaps generated by cleavage on both sides of the 5' end. In addition, non-specific amplification products formed between the primers and the template will also be cleaved to varying degrees (Figure 1). Therefore, under the action of the enzyme that specifically cleaves nucleotides containing U bases, not only are the excess primers digested, but non-specific amplification products containing primers are also eliminated to a certain extent. It should be noted that the inventors modified the primers with U bases and then digested them with an enzyme that specifically cuts nucleotides containing U bases. Different primer modifications require different types of enzymes for specific single-base cutting, but all achieve the goal of cleaving the modified sites through the enzyme and then digesting the primers. These single-base gaps will affect the second round of PCR amplification and must be repaired before the second round of PCR. Otherwise, the universal sequences at both ends will be lost, making them unable to complement the second-round tag primers, leading to amplification failure. Therefore, the second problem to be solved is repairing the gaps in the target amplification product, which cannot be ligated using conventional ligases. Currently, no commercial reagent claims to be able to repair single-base gaps in double-stranded DNA. The inventors of the present disclosure creatively used Taq DNA polymerase, which has both strong 5'-3' exonuclease activity and DNA polymerization activity. This enzyme has very strong 5'-3' exonuclease activity and has good affinity for double-stranded DNA with single-base gaps. Enzymes known in the art that have both polymerase activity and 5'-3' exonuclease activity are suitable for the present disclosure, and are not limited to Taq DNA polymerase.

[0076] According to some specific embodiments of the present disclosure, the present disclosure proposes a nested PCR method, the method comprising:

[0077] (a) performing a first round of PCR on the sample to be tested using a first primer with a single-base restriction site to obtain a first round of PCR product;

[0078] (b) adding enzyme 1, enzyme 2, and a tag primer to the first-round PCR product to perform a second-round PCR reaction;

[0079] Among them, the enzyme 1 is used to cut the sequence with a single-base restriction site, and the enzyme 2 is used to repair the single-base gap formed in the first-round PCR product after being cut by the enzyme 1. The sequence with a single-base restriction site cut by enzyme 1 includes the first primer pair, the specific PCR product in the first-round PCR product, and the non-specific PCR product.

[0080] According to a specific embodiment of the present disclosure, in the first round of PCR, the first primer pair is a set of specific primers containing U bases, which are used to perform PCR amplification on the extracted sample nucleic acid (e.g., nucleic acid extracted from a clinical sample for pathogen detection). The number of primer pairs used depends on the number of targets to be detected, and at least one primer pair is required for each target.

[0081] According to a specific embodiment of the present disclosure, the primers in the first primer pair are composed of an 18-24nt target-specific sequence (Specific sequence) and a 22nt universal sequence (Universal sequence). The target-specific sequence here is a sequence complementary to a portion of the nucleic acid sequence of the sample to be detected, and the universal sequence can be designed and changed according to different sequencing platforms or sequencing purposes. It is only necessary to ensure that the universal sequence is complementary to a portion of the tag primer sequence. For example, the universal sequence US-a: 5'-TTTCTGTTGGTGCTGATATTGC-3' (SEQ ID NO: 1) is added to the 5' end of the forward primer, and the universal sequence US-b: 5'-ACTTGCCTGTCGCTCTATCTTC-3' (SEQ ID NO: 2) is added to the 5' end of the reverse primer.

[0082] According to a specific embodiment of the present disclosure, U bases are incorporated into the primers in the first primer pair, and U bases are used to replace some T bases in the primers when synthesizing the primers. Each primer needs to incorporate 2 U bases, one at a fixed position on the universal sequence, and the other in the target-specific sequence region, which can be designed at a position 10-15nt away from the first U base. The total length of the primers is between 40-45nt.

[0083] For example, the original sequence of primer S.pne-F1 is:

[0084] 5'-TTTCTGTTGGTGCTGATATTGCGGTGAGAGATGTCATTCCGATTAA-3' (SEQ ID NO: 3),

[0085] After U base modification:

[0086] 5'-TTTCTGTTGGTGCTGAUATTGCGGTGAGAGAUGTCATTCCGATTAA-3' (SEQ ID NO: 4),

[0087] The original sequence of primer S.pne-R1 is:

[0088] 5'-ACTTGCCTGTCGCTCTATCTTCGGCAACAGCATCTTGTATACC-3' (SEQ ID NO: 5),

[0089] After modification with U base:

[0090] 5'-ACTTGCCTGTCGCTCTAUCTTCGGCAACAGCAUCTTGTATACC-3' (SEQ ID NO: 6).

[0091] According to a specific embodiment of the present disclosure, a single or continuous modified nucleotide is incorporated into the primer in the first primer pair, for example, 1-5 consecutive nucleotides on the primer are any one of uracil deoxyribonucleotide, 5-hydroxyuracil deoxyribonucleotide, 5-hydroxymethyluracil deoxyribonucleotide, and 5-formyluracil deoxyribonucleotide. Preferably, the number of consecutive modified nucleotides is 1-3, and one primer can have one or more single or continuous modified nucleotides.

[0092] According to a specific embodiment of the present disclosure, discontinuous modified nucleotides are incorporated into the primers in the first primer pair, for example, a nucleotide of any one of uracil deoxyribonucleotide, 5-hydroxyuracil deoxyribonucleotide, 5-hydroxymethyluracil deoxyribonucleotide, and 5-formyluracil deoxyribonucleotide is incorporated every 10-15 nt.

[0093] According to a specific embodiment of the present disclosure, after the first primer pair is cleaved by the enzyme 1, the unreacted first primer and a portion of the nucleic acid in the nonspecific product exist as single strands. After the U base is removed, the fragments are fragmented and still exist in the second-round PCR reaction system. However, due to their short fragments, they are difficult to bind to the template at conventional annealing temperatures. Preferably, each cleaved fragment is no longer than 18 bp to ensure that in the next PCR step, the shorter first primer fragments are difficult to bind to the template, thereby achieving the true removal of excess primers.

[0094] In some embodiments, the tag primers are used to distinguish different sources of the samples to be tested.

[0095] In some embodiments, the enzyme 1 is mixed with the enzyme 2 and the tag primer and then added or added sequentially to the first-round PCR product. The main purpose of the enzyme 1 is to digest the incomplete reaction of the first primer pair and some non-specific amplification products in the first-round PCR reaction. The main purpose of the enzyme 2 is to repair the single-base gap generated at the 5' end on both sides of the target amplification product.

[0096] In some specific embodiments, the second-round PCR reaction includes a first incubation reaction and a second incubation reaction. The second incubation is performed after the first incubation reaction is completed. The main purpose of the first incubation reaction is to digest the first primer and some non-specific amplification products that were not completely reacted in the first-round PCR reaction. The main purpose of the second incubation reaction is to repair the single-base gaps generated at the 5' ends on both sides of the target amplification product. The two incubation reactions can be performed in the same system, that is, enzyme 1 and enzyme 2 are added to the first-round PCR product together, and the first incubation reaction and the second incubation reaction are performed in sequence by controlling the reaction temperature of enzyme 1 and enzyme 2. Alternatively, enzyme 1 can be added alone for the first incubation reaction, and then enzyme 2 can be added for the second incubation reaction after the first incubation reaction. The tag primer can be added during the incubation reaction, or the tag primer can be added after the incubation reaction is completed.

[0097] According to a specific embodiment of the present disclosure, the enzyme 1, enzyme 2, and label primer can be pre-mixed and added to the first-round PCR product for incubation to save operation time and reduce the risk of sample cross-contamination.

[0098] In some specific embodiments, the first incubation reaction time is not less than 5 minutes, preferably 5-25 minutes, and more preferably 10-15 minutes. When the first incubation reaction time is not less than 5 minutes, the USER enzyme has a good removal effect on the primer.

[0099] In some specific embodiments, the USER enzyme content is 100-300 U / mL and the incubation condition is 20-45°C for 10-15 min, which has a good removal effect on the primer. Preferably, the USER enzyme content is 200 U / mL and the incubation condition is 37°C for 10-15 min.

[0100] In some specific embodiments, the second incubation time is 1-25 min, preferably 5-20 min, and more preferably 5-10 min. When the first incubation time is 1-25 min, Taq DNA polymerase can effectively repair the 5' end gaps on both sides of the target amplification product, thereby achieving the second round of PCR amplification.

[0101] In some specific embodiments, under the experimental conditions of 200-400 U / mL of Taq DNA polymerase and 65-72° C. for 5-10 min, the 5' end gaps on both sides of the target amplification product can be well repaired, thereby achieving a second round of PCR amplification.

[0102] In some specific embodiments, the index primer consists of a tag sequence (Barcode sequence) and a reverse complementary sequence of the above-mentioned universal sequence, wherein the tag sequence is close to the 5' end. The index primer can be directly obtained from a commercial kit (EXP-PBC096, Nanopore), which contains 96 different index primers from BC01 to BC96 and can be used simultaneously for mixed sample library construction of up to 96 samples. The index primer can also be obtained by sequence synthesis, and is not limited to the number of samples for mixed sample library construction.

[0103] In some specific embodiments, the number of amplification cycles in the first round of PCR is generally set to 15-25 cycles, and a small amount of target amplification product is first obtained at a lower cycle number, which serves as a template for the second round of PCR amplification; the second round of PCR uses the small amount of target amplification product obtained in the first round of PCR as a template, and the number of amplification cycles is generally set to 12-15 cycles. While adding sample tags to the target amplification products, tag primers containing universal sequences are used to enrich them to obtain a sufficient amount of library for sequencing.

[0104] Based on a nested PCR method proposed in the present disclosure, the present disclosure further proposes a library construction method suitable for targeted detection, the method comprising:

[0105] (c) obtaining the second-round PCR product by nested PCR;

[0106] (d) Connecting the second-round PCR products to sequencing adapters to obtain a sequencing library.

[0107] In some specific embodiments, the purification includes purifying the second-round PCR product using any method for purifying DNA known in the art, such as a magnetic bead method, a column method, and the like.

[0108] According to a specific embodiment of the present disclosure, the purification process includes adding magnetic beads to the second-round PCR product, mixing, and then incubating. The magnetic beads are then washed at least twice with ethanol, excess ethanol removed, and air-dried. Elution is then performed with nuclease-free water, and the eluted product is recovered. Because the amplified products of the second-round PCR are each labeled with a different sample, even if PCR-generated aerosols are introduced into individual samples during the purification process, cross-contamination of the samples will not occur, as each sample's label sequence is unique, and any other sample that has been mixed can be distinguished through sequencing.

[0109] In another aspect, the present disclosure provides a library suitable for sequencing, wherein the library is prepared by the above-mentioned library construction method.

[0110] In some embodiments, the library is suitable for sequencing by synthesis and nanopore sequencing methods.

[0111] In some specific embodiments, the sequencing by synthesis includes second-generation sequencing and third-generation sequencing, and the sequencing platform of the second-generation sequencing includes rolling circle amplification sequencing, bridge amplification sequencing, and single-molecule sequencing.

[0112] In some specific embodiments, in order to adapt to the long-read sequencing characteristics of the nanopore sequencing platform and take into account the amplification efficiency, the length of the amplification product can be set between 500-1000bp, which can not only obtain a library length significantly higher than that of second-generation sequencing, but also obtain good amplification efficiency and shorter amplification time.

[0113] In some specific embodiments, the present disclosure provides an improved two-step PCR library construction method for pathogen-targeted detection. This method utilizes the nuclease cleavage activity of the USER enzyme, combined with the DNA polymerization activity and 5'-3' exonuclease activity that Taq DNA polymerase may possess simultaneously, to establish a novel primer removal method that replaces the purification step between the first and second rounds of PCR. This method successfully resolves the problem of cross-contamination between samples caused by the purification operation. Furthermore, this method can effectively remove nonspecific amplification products, improve the specificity of targeted amplification, and simplify the experimental process. This method is an efficient, convenient, and cross-contamination-avoiding library construction method suitable for nanopore sequencing platforms.

[0114] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0115] The primers used in the following examples were all synthesized by Shanghai Sangon Biotechnology Co., Ltd. The library construction kit SQK-LSK110 and sample label expansion package EXP-PBC096 used were purchased from Oxford Nanopore. The PCR amplification reagents used were purchased from Roche, USER enzyme and T4 DNA ligase were purchased from NEB, nuclease-free water was purchased from Invitrogen, and the magnetic beads used were Beckman's AMPure XP magnetic beads. The pathogenic nucleic acids used were all purchased from Beina Biotechnology. The sequencer used was Oxford Nanopore's MinION Mk1C, and the sequencing chip type was FLO-MIN106.

[0116] Example 1 Test of primer digestion effect of USER enzyme

[0117] In this example, different USER enzyme concentrations and reaction times were set to perform primer digestion tests on the amplification products of pathogenic multi-target nucleic acid reference products.

[0118] The specific implementation process is as follows:

[0119] (1) Preparation of pathogenic multi-target nucleic acid reference samples: The nucleic acids of the samples in the following table were used to prepare reference samples according to the nucleic acid copy numbers described in Table 1:

[0120] Table 1

[0121] (2) Preparation of primer mixture for the first round of PCR amplification: A total of 10 pairs of primers were designed to perform multiplex PCR amplification on the five targets in the reference product, with two pairs of primers for each target. Each primer incorporated two U bases. The primer sequences are shown in Table 2:

[0122] Table 2

[0123] Among them, S.pne is Streptococcus pneumoniae, A.bau is Acinetobacter baumannii, P.aer is Pseudomonas aeruginosa, E.col is Escherichia coli, and K.pne is Klebsiella pneumoniae.

[0124] (3) PCR reaction: Prepare the PCR reaction system according to Table 3:

[0125] Table 3

[0126] Vortex the prepared reaction system, centrifuge briefly, and place in a PCR instrument to perform the reaction according to the program in Table 4:

[0127] Table 4

[0128] (4) Primer digestion: Different amounts of USER enzyme were added to the above PCR products, and primer digestion reactions were performed according to the groups in Table 5:

[0129] Table 5

[0130] (5) Perform gel electrophoresis on the products of each digestion system using 1% agarose gel to check the primer residue;

[0131] (6) Result detection: According to the results of agarose gel electrophoresis, both digestion system 6 and digestion system 9 can achieve good primer digestion effect (Figure 2), that is, at an enzyme content of 10U and a reaction time of 10-15min at 37°C, the residual primers in the first-round PCR products can be successfully digested.

[0132] Example 2 Screening of Taq DNA polymerase

[0133] To obtain a Taq DNA polymerase capable of repairing gaps in the target amplified product, this paper screened and tested Taq DNA polymerases from multiple brands. The results showed that, after primer digestion with USER enzyme, a 5-minute DNA chain extension reaction using Taq DNA polymerase successfully repaired gaps on both sides of the 5' end of the target amplified product.

[0134] The specific implementation process is as follows:

[0135] (1) First, refer to steps (1)-(4) in Example 1 to obtain a PCR product after USER enzyme digestion. The selected primer digestion experimental conditions are: add 10U USER enzyme to 50uL of amplified product and react at 37°C for 10min. Thus, a PCR product after primer digestion is obtained;

[0136] (2) 10 U of DNA polymerases of different brands were added to the reaction product of step (1). The models and brands of DNA polymerases tested are shown in Table 6:

[0137] Table 6

[0138] (3) Then add 2 μL of the tag primer (EXP-PBC096 kit, Nanopore), pipette to mix, and centrifuge briefly;

[0139] (4) Carry out PCR reaction according to the procedure in Table 7:

[0140] Table 7

[0141] (5) Perform gel electrophoresis on the products of each test group using 1% agarose gel to check the amplification of the target product;

[0142] (6) Result detection: According to the results of agarose gel electrophoresis, only test enzyme 6 can obtain the ideal second-round PCR amplification product (Figure 3), that is, after gap repair by Tiangen High Affinity HotStart Taq, the second-round PCR amplification can be successfully achieved.

[0143] The inventors speculate that although Taq DNA polymerase is essentially used for the replication of nucleic acid chains, different Taq DNA polymerases have undergone different mutational transformations and chemical modifications. Some mutation screening methods focus on improving the DNA polymerization activity of the enzyme while ignoring or even sacrificing its 5'-3' exonuclease activity. The Tiangen High Affinity HotStart Taq obtained by screening in the present disclosure just has the enzyme activity that meets the requirements of this scheme, and therefore can successfully achieve the gap repair of the target product. It should be noted that this experiment only screened 9 types of enzymes, which does not mean that those skilled in the art cannot screen out other enzymes like Tiangen High Affinity HotStart Taq that retain higher DNA polymerase activity and 5'-3' exonuclease activity.

[0144] Example 3 Optimization test of Taq DNA polymerase addition amount and reaction time

[0145] After obtaining Taq DNA polymerase for gap repair in first-round PCR products, the present invention optimized the enzyme dosage and reaction time required for the gap repair process. These tests revealed that an enzyme dosage between 10 and 20 units and a reaction time of 5 to 10 minutes at 72°C resulted in optimal gap repair results. Specifically, under these reaction conditions, ideal second-round PCR amplification products were obtained.

[0146] The specific implementation process is as follows:

[0147] (1) First, refer to steps (1)-(4) in Example 1 to obtain a PCR product after USER enzyme digestion. The selected primer digestion experimental conditions are: add 10U USER enzyme and react at 37°C for 10 minutes. Thus, a PCR product after primer digestion is obtained;

[0148] (2) Different amounts of Tiangen Taq DNA polymerase were added to the reaction product of step (1). The amount of enzyme added and the reaction time of each reaction system are shown in Table 8:

[0149] Table 8

[0150] (3) Then add 2 μL of the tag primer (EXP-PBC096 kit, Nanopore), pipette to mix, and centrifuge briefly;

[0151] (4) Perform PCR reaction according to the procedure in Table 9:

[0152] Table 9

[0153] (5) Perform gel electrophoresis on the products of each test group using 1% agarose gel to check the amplification of the target product;

[0154] (6) Result detection: According to the results of agarose gel electrophoresis, repair systems 2, 3, 5, and 6 can all obtain ideal second-round PCR amplification products (Figure 4). That is, when the enzyme addition amount is 10-20U and the reaction is carried out at 72°C for 5-10 minutes, ideal gap repair effects can be obtained.

[0155] Example 4 Detection of pathogenic microorganism samples

[0156] In this example, the targeted library construction method provided by the present disclosure was used to construct libraries for five pathogen multi-target positive reference samples containing five pathogen targets and one negative reference sample. The libraries were sequenced using the Nanopore sequencing platform to analyze the target detection and cross-contamination between samples.

[0157] The specific implementation process is as follows:

[0158] (1) Preparation of nucleic acid references: The nucleic acids of the samples listed in the following table were used to prepare references according to the nucleic acid copy numbers listed in Table 10:

[0159] Table 10

[0160] (2) Preparation of primer mixture for the first round of PCR amplification: A total of 42 pairs of primers were designed to perform multiplex PCR amplification on 21 targets in the reference product, with 2 pairs of primers for each target. Each primer incorporated 2 U bases. The primer sequences are shown in Table 11:

[0161] Table 11

[0162] Among them, S.pne is Streptococcus pneumoniae, A.bau is Acinetobacter baumannii, P.aer is Pseudomonas aeruginosa, E.col is Escherichia coli, K.pne is Klebsiella pneumoniae, E.fae2 is Enterococcus faecium, S.mar is Serratia marcescens, S.epi is Staphylococcus epidermidis, S.aga is Streptococcus agalactiae, S.mal is Stenotrophomonas maltophilia, E.fae is Enterococcus faecalis, K.oxy is Klebsiella oxytoca, S.pyo is Streptococcus pyogenes, H.inf is Haemophilus influenzae, S.aur is Staphylococcus aureus, P.mir is Proteus mirabilis, S.mit is Streptococcus mitis, L.mon is Listeria monocytogenes, E.clo is Enterobacter cloacae, H.par is Haemophilus parainfluenzae, and P.flu is Pseudomonas fluorescens.

[0163] (3) First round PCR reaction: Prepare the PCR reaction system according to Table 12:

[0164] Table 12

[0165] Vortex the prepared reaction system, centrifuge briefly, and place in a PCR instrument to perform the reaction according to the program in Table 13:

[0166] Table 13

[0167] (4) Second round of PCR reaction: 10 U of USER enzyme, 10 U of Taq DNA polymerase (Tiangen) and 2 μL of tag primer (EXP-PBC096 kit, Nanopore) were directly added to the above amplified product, shaken to mix, centrifuged briefly, and the second round of PCR reaction was carried out according to the procedure in Table 14:

[0168] Table 14

[0169] (5) PCR product purification: Add 0.7 times the volume of magnetic beads to the second-round PCR product, mix well, and incubate for 5 minutes. Wash the magnetic beads at least twice with 80% ethanol. After washing, remove excess ethanol and let them dry for at least 1 minute. Then, add 20-30 μL of nuclease-free water for elution and recover the eluted product.

[0170] (6) Nucleic acid concentration determination: quantifying the nucleic acid in the product recovered in step (5);

[0171] (7) Library mixing: Libraries with different sample labels are mixed in equal molar amounts;

[0172] (8) Adapter ligation: Add the mixed library according to Table 15, flick the tube to mix between each addition, and finally mix thoroughly with a pipette:

[0173] Table 15

[0174] Incubate at room temperature for 10 minutes. Add 40uL AMPure XP magnetic beads to the system, mix well and incubate at room temperature for 5 minutes. Discard the supernatant after magnetic separation and wash twice with 250uL short fragment buffer (SFB). Open the lid and dry for 30 seconds. Add 15uL elution buffer (EB), centrifuge and incubate at room temperature for 10 minutes. Magnetic separation for more than 1 minute, recover 13uL eluate into a new 1.5mL EP tube. The resulting library can be directly sequenced on the nanopore sequencer;

[0175] (9) Sequencing: The library was sequenced using a Nanopore MinION 1kC sequencer. The sequencing parameters were set as follows: sequencing time was set to 1 h, Q value filter threshold was Q8, and read length filter threshold was set to 200 bp.

[0176] (10) Data Analysis: After the data is downloaded, the sample will be automatically split according to the sample tag sequence, and the sequencing sequence will be filtered according to the read length and quality value. The filtered sequence will undergo downstream bioinformatics analysis and be compared with the pathogen database to obtain the number of detected sequences for each target in each sample.

[0177] Comparative Example 1 Two-step PCR library construction method for detection of pathogenic microorganism samples

[0178] In this example, the original targeted library construction method was used to construct libraries for five pathogen multi-target positive reference samples containing five pathogen targets and one negative reference sample. The libraries were sequenced using the Nanopore sequencing platform to analyze the target detection and cross-contamination between samples.

[0179] The specific implementation process is as follows:

[0180] (1) Preparation of nucleic acid reference material: The reference material used in this example is exactly the same as the reference material used in Example 4;

[0181] (2) Preparation of primer mixture for the first round of PCR amplification: A total of 42 pairs of primers were designed to perform multiplex PCR amplification on 21 targets in the reference product, with 2 pairs of primers for each target. No U base was incorporated into the primer sequence, and the remaining sequence was completely consistent with the primer sequence in Example 4. The primer sequences are shown in Table 16:

[0182] Table 16

[0183] Among them, S.pne is Streptococcus pneumoniae, A.bau is Acinetobacter baumannii, P.aer is Pseudomonas aeruginosa, E.col is Escherichia coli, K.pne is Klebsiella pneumoniae, E.fae2 is Enterococcus faecium, S.mar is Serratia marcescens, S.epi is Staphylococcus epidermidis, S.aga is Streptococcus agalactiae, S.mal is Stenotrophomonas maltophilia, E.fae is Enterococcus faecalis, K.oxy is Klebsiella oxytoca, S.pyo is Streptococcus pyogenes, H.inf is Haemophilus influenzae, S.aur is Staphylococcus aureus, P.mir is Proteus mirabilis, S.mit is Streptococcus mitis, L.mon is Listeria monocytogenes, E.clo is Enterobacter cloacae, H.par is Haemophilus parainfluenzae, and P.flu is Pseudomonas fluorescens.

[0184] (3) First round PCR reaction: Prepare the PCR reaction system according to Table 17:

[0185] Table 17

[0186] Vortex the prepared reaction system, centrifuge briefly, and place in a PCR instrument to perform the reaction according to the program in Table 18:

[0187] Table 18

[0188] (4) Purification of the first-round PCR product: Add 0.7 times the volume of magnetic beads to the first-round PCR product, mix well, and incubate for 5 minutes. Wash the magnetic beads at least twice with 80% ethanol. After washing, remove excess ethanol and let them dry for at least 1 minute. Then, add 20-30 μL of nuclease-free water for elution and recover the eluted product.

[0189] (5) Second round of PCR reaction: Prepare the PCR reaction system according to Table 19:

[0190] Table 19

[0191] Vortex the prepared reaction system, centrifuge briefly, and place in a PCR instrument to perform the reaction according to the program in Table 20:

[0192] Table 20

[0193] (6) PCR product purification: Add 0.7 times the volume of magnetic beads to the second-round PCR product, mix well, and incubate for 5 minutes. Wash the magnetic beads at least twice with 80% ethanol. After washing, remove excess ethanol and let them dry for at least 1 minute. Then, add 20-30 μL of nuclease-free water for elution and recover the eluted product.

[0194] (7) Nucleic acid concentration determination: quantifying the nucleic acid in the product recovered in step (5);

[0195] (8) Library mixing: Libraries with different sample labels are mixed in equal molar amounts;

[0196] (9) Adapter ligation: Add the mixed library according to Table 21, flick the tube to mix between each addition, and finally mix thoroughly with a pipette:

[0197] Table 21

[0198] Incubate at room temperature for 10 minutes. Add 40 μL of AMPure XP magnetic beads to the system, mix thoroughly, and incubate at room temperature for 5 minutes. Perform magnetic separation and discard the supernatant. Wash twice with 250 μL of Short Fragment Buffer (SFB). Uncap and dry for 30 seconds. Add 15 μL of Elution Buffer (EB), centrifuge, and incubate at room temperature for 10 minutes. Perform magnetic separation for 1 minute or longer, and collect 13 μL of the eluate into a new 1.5 mL EP tube. The resulting library can be directly sequenced on a nanopore sequencer.

[0199] (10) Sequencing: The library was sequenced using a Nanopore MinION 1kC sequencer. The sequencing parameters were set as follows: sequencing time was set to 1 h, Q value filter threshold was Q8, and read length filter threshold was set to 200 bp;

[0200] (11) Data Analysis: After the data is downloaded, the sample will be automatically split according to the sample tag sequence, and the sequencing sequence will be filtered according to the read length and quality value. The filtered sequence will undergo downstream bioinformatics analysis and be compared with the pathogen database to obtain the number of detected sequences for each target in each sample.

[0201] Result detection

[0202] In Example 4 and Comparative Example 1, the number of detected sequences for each pathogen target in the reference sample was analyzed. For better comparison, the number of detected sequences was normalized and the RPT (Reads Per Ten Thousands) indicator was set, that is, the number of pathogen target sequences detected per 10,000 sequencing reads.

[0203] The detection results of each positive reference sample in Example 4 and Comparative Example 1 are shown in Table 22. The test results show that the pathogen target in each positive reference sample was successfully detected in both Example 4 and Comparative Example 1, indicating that the nested PCR method provided by the present disclosure can be used for sequencing library construction and achieve targeted detection of pathogens. In addition, compared with Comparative Example 1, the RPT sequence values ​​detected in the targets in each positive reference sample in Example 4 were higher than those in Comparative Example 1, indicating that the nested PCR method provided by the present disclosure has higher target amplification specificity than the original two-step PCR method.

[0204] In addition, this test analyzed the contamination rate of each sample. The contamination rate was calculated as follows: for a given sample, contamination rate = number of non-self-target sequences detected / total number of sequences in the sample. The contamination rates of the reference samples in Example 4 and Comparative Example 1 are shown in Table 23. As can be seen from this table, the nested method provided by this disclosure reduces the cross-contamination rate by over 60-fold compared to the conventional two-step PCR method, significantly improving cross-contamination between samples in pathogen detection and enhancing the accuracy of pathogen detection.

[0205] Table 22

[0206] Table 23

[0207] Results showed that the library construction method provided by this disclosure reduced the contamination rate between samples by more than 60 times compared to the existing two-step PCR library construction method. This is very important for the accuracy of detection of negative samples and samples containing low-abundance pathogens.

[0208] The disclosed embodiments utilize the enzymatic cleavage characteristics of USER enzyme and Taq DNA polymerase to establish a new primer removal method, and based on this, provide a new library construction method suitable for the nanopore platform, which not only simplifies the experimental process, but also significantly improves the problem of cross-contamination between samples and improves the accuracy of pathogenic sample detection.

[0209] The disclosed solution uses USER enzyme to digest primers while also removing some nonspecific amplification products, thereby improving the specificity of target amplification and ensuring successful detection of pathogenic targets. It only requires adding the enzyme and label primers directly to the first-round PCR products and adding an additional 15 minutes of reaction time before the second-round PCR reaction procedure. However, it eliminates the tedious nucleic acid purification step, shortening the entire process to 2.5 hours, making it a simpler and faster library construction method.

[0210] The present disclosure provides a method for removing excess primers in nested PCR, and based on this, provides a new targeted library construction method suitable for nanopore platforms. The technical core involved in this method is to first modify the amplification primers specifically, and then use the different characteristics of the enzymes to achieve the digestion of residual primers in the first round of PCR amplification and the repair of the gap in the target amplification product. In the embodiments of the present disclosure, the inventors used the single-base cleavage characteristics of the USER enzyme, the DNA polymerase activity and 5'-3' exonuclease activity of the Taq DNA polymerase respectively. It should be noted that any enzyme with a single-base cleavage characteristic can be used as enzyme 1 in the present disclosure. For enzymes with different single-base cleavage characteristics, it is only necessary to change the enzyme cleavage site on the first primer in the present disclosure accordingly to achieve the same or similar experimental results as those in the present disclosure. Similarly, any enzyme with both strong DNA polymerase activity and 5'-3' exonuclease activity can also be used as enzyme 2 in the present disclosure. Any combination of enzymes with single-base cleavage properties, DNA polymerase activity, and 5'-3' exonuclease activity to achieve the purpose of digesting residual primers and repairing gaps in target amplification products should be considered to be the same as the technical principles of the present disclosure and fall within the scope of protection of this invention.

[0211] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and features of different embodiments or examples, unless they are mutually inconsistent.

[0212] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A nested PCR method, characterized in that: The method comprises: (a) performing a first round of PCR on the sample to be tested using a first primer with a single-base restriction site to obtain a first round of PCR product; (b) adding enzyme 1, enzyme 2, and label primer to the first round PCR product to carry out a second round of PCR reaction; The enzyme 1 is used to cut the sequence with a single-base restriction site, and the enzyme 2 is used to repair the single-base gap formed by the cutting of the enzyme 1 in the first-round PCR product.

2. The method according to claim 1, characterized in that: Any one primer in the first primer pair includes: a targeting specific sequence and a universal sequence, the targeting specific sequence is complementary to a portion of the nucleic acid sequence of the sample to be tested, and the universal sequence is complementary to a portion of the sequence of the tag primer.

3. The method according to claim 2, characterized in that The tag primer includes a tag sequence and a sequence complementary to the universal sequence.

4. The method according to claim 2, characterized in that: The targeting specific sequence contains the single-base restriction site.

5. The method according to claim 2, characterized in that: The universal sequence contains the single-base restriction site.

6. The method according to claim 2, characterized in that The single-base restriction site is contained in both the targeting specific sequence and the universal sequence.

7. The method according to claim 2, characterized in that The targeting specific sequence and the universal sequence each contain at least one single-base restriction site.

8. The method according to claim 2, characterized in that: The sequence length of any one primer in the first primer pair is 36-48 nt.

9. The method according to claim 1, characterized in that: Any one primer in the first primer pair contains at least one single-base restriction site.

10. The method according to claim 1, characterized in that Any one primer in the first primer pair contains 1-5 restriction enzyme cutting sites.

11. The method according to claim 1, characterized in that: Any one primer in the first primer pair contains two restriction enzyme cutting sites.

12. The method according to claim 1, characterized in that The single-base restriction site includes modified ribonucleotides or deoxyribonucleotides.

13. The method according to claim 1, characterized in that The single-base restriction site includes at least one of uracil deoxyribonucleotide, 5-hydroxyuracil deoxyribonucleotide, 5-hydroxymethyluracil deoxyribonucleotide, and 5-formyluracil deoxyribonucleotide.

14. The method according to claim 1, characterized in that The enzyme 1 comprises at least one of USER enzyme, UDG enzyme, hsMUG1 enzyme and lytic enzyme Endo VIII.

15. The method according to claim 1, characterized in that The enzyme 2 has both DNA polymerase activity and 5'-3' exonuclease activity.

16. The method according to claim 1, characterized in that The enzyme 2 includes Taq DNA polymerase or DNA polymerase I.

17. The method according to claim 1, characterized in that In step (b), the second round of PCR reaction includes two incubation reactions and PCR reactions, wherein the first incubation reaction includes using enzyme 1 to cut the sequence with a single-base restriction site, and the second incubation reaction includes using enzyme 2 to repair the single-base gap formed in the first round of PCR product after being cut by enzyme 1.

18. The method according to claim 1, characterized in that Step (b) comprises the following steps: Enzyme 1, enzyme 2 and label primer were sequentially added to the first round PCR product to carry out the second round PCR reaction.

19. The method according to claim 1, characterized in that Step (b) comprises the following steps: Enzyme 1, enzyme 2 and label primer are mixed and added to the first round PCR product to carry out the second round PCR reaction.

20. The method according to claim 1, characterized in that Step (b) comprises the following steps: After adding enzyme 1 and enzyme 2 to the first round PCR product for two incubation reactions, the label primer is added to carry out PCR reaction.

21. The method according to claim 17, characterized in that The temperature of the first incubation reaction is different from the temperature of the second incubation reaction.

22. The method according to claim 17, characterized in that The time of the first incubation reaction is not less than 5 minutes, and the temperature of the first incubation reaction is 20-45°C.

23. The method according to claim 17, characterized in that The second incubation reaction time is 1-25 minutes, and the second incubation reaction temperature is 65°C-72°C.

24. Use of the nested PCR method according to any one of claims 1 to 23 in library construction.

25. A method for constructing a sequencing library, characterized in that: The construction method comprises: (c) obtaining the second round of PCR products using nested PCR according to any one of claims 1 to 23; (d) Connecting the second round PCR products to a sequencing adapter to obtain a sequencing library.

26. Use of the construction method according to claim 25 in sequencing.

27. A library suitable for sequencing, characterized in that The library is prepared by the construction method described in claim 25.

28. The library according to claim 27, characterized in that The library is suitable for sequencing by synthesis and nanopore sequencing.

29. A library construction kit, comprising: Reagents configured to construct the library of claim 27.

30. The library construction kit according to claim 29, characterized in that: The reagent includes at least one of a primer and an enzyme.

31. Use of the library construction kit according to claim 29 in nanopore targeted detection.

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