WO2024120262A1 - Cleavable ring-like primer, kit thereof, and amplification method therefor - Google Patents

Abstract

The present invention provides a cleavable ring-like primer and a kit thereof, and further provides a corresponding PCR amplification method and a preparation method for the cleavable ring-like primer. The cleavable ring-like primer comprises a universal primer sequence and a target specific sequence, and the target specific sequence comprises at least one ribonucleotide. The cleavable ring-like primer designed by the present invention does not form dimers, can increase the amplification efficiency of a specific target, has increased stability, and thus can increase amplification stability.

Description

A cleavable circular primer and its kit and amplification method

CROSS-REFERENCE TO RELATED APPLICATIONS

The present disclosure claims priority to Chinese patent application No. 202211571299.7, filed with the State Intellectual Property Office of China on December 8, 2022, and entitled “A cleavable circular primer, a kit and an amplification method thereof”, the entire contents of which are incorporated into the present disclosure by reference.

Technical Field

The present invention relates to the field of biological detection, and in particular to a cleavable circular primer and a kit and an amplification method thereof.

Background technique

Gene sequencing plays a very important role in the research of life sciences, and high-throughput sequencing technology is one of the most important technologies in the current sequencing field. To perform high-throughput sequencing on sample sequences, it is first necessary to construct a DNA library that can be used for sequencing. The traditional shotgun sequencing library construction process has a long process, which generally requires the following steps: (1) Use ultrasound to break the genomic DNA molecules to be tested into sequence fragments of 200 to 500 bp in length; (2) Fill the ends of the broken genome; (3) Add an A tail to the DNA fragments after filling; (4) Connect the fragments with A tails; (5) PCR amplify the DNA fragments after connecting the adapters; (6) Purify the amplified library with magnetic beads. Traditional shotgun DNA high-throughput sequencing targets whole genome DNA, which contains a large number of non-target gene sequences. These non-target sequences seriously interfere with the analysis of target sequences and even make the target region unable to be sequenced. For example, in tumor gene mutation detection and pathogenic microorganism detection, more than 95% of gene sequences are useless sequences, and shotgun genomic DNA sequencing cannot achieve the detection purpose.

In order to reduce the interference of non-target sequences in sequencing, the industry has developed high-throughput targeted sequencing technology (tNGS). The specific process of high-throughput targeted sequencing is to design capture primers or probes for the target detection area, such as tumor hot spot mutations, pathogenic microorganisms of a certain syndrome, etc., to capture the target sequence fragments from the massive whole genome sequence fragments, and add sequencing-capable adapters to both ends of the captured gene sequence fragments to form a targeted DNA sequencing library for bioinformatics analysis. Since only the target region sequence can be tested, the amount of sequencing data can be one thousandth or even one ten-thousandth of conventional genome sequencing. Targeted sequencing technology greatly saves data volume costs and can significantly improve detection performance. The general process of high-throughput targeted sequencing is: (1) Design capture probes for the target sequence region; (2) Construct a genomic DNA library; (3) Use capture probes to hybridize and capture the target region sequence from the DNA library; (4) Amplify the captured target region sequence to obtain a capture library that can be used for sequencing.

Compared with shotgun genomic DNA sequencing, targeted sequencing has great advantages in sequencing the target region sequence, but the entire capture library preparation process often takes 8 to 16 hours, the process is cumbersome, and the time and economic costs are high. These shortcomings limit the further application of targeted sequencing technology. Another newly developed targeted sequencing library construction method, the multiplex amplification method, perfectly solves the above problems. The multiplex amplification method can be designed by designing specific amplification primers in the target sequence, and adding adapter tags that can be used for sequencing at both ends of the specific primers. Multiple amplification enriches the target region and adds sequencing adapters. After two rounds of PCR amplification and purification, the targeted sequencing library can be obtained. This method has begun to be widely used in many fields such as agriculture, soil, forestry, microbiology, and human medicine.

Although the multiplex amplification method is simpler than the traditional library construction process, it requires tube transfer, magnetic bead purification, second round amplification or other operations such as adding sequencing adapters after the first round of targeted amplification. During the tube transfer and purification process after the first round of targeted amplification, aerosol contamination is very easy to occur, resulting in cross-contamination of other samples and may cause false positives in the final test results. In addition to being prone to aerosol contamination, the current multiplex PCR library construction methodology has extremely high requirements for the design of specific targeted primers. Specific requirements include: each specific primer cannot form primer dimers by itself or with each other; each pair of primers is required to have one and only one specific targeting region; each primer cannot have more than 3 consecutive identical bases at the 3' end. With such stringent primer design conditions, it will be very difficult to design multiple primers for super-multiple targeted amplification library construction, which often has hundreds or even thousands of primers.

In the field of pathogen detection, due to the wide variety of pathogens and the high sequence similarity of subspecies and bacterial groups, there are higher requirements for multiplex PCR primer design. In clinical samples, pathogen nucleic acids are generally accompanied by interference from most host nucleic acids, which further increases the difficulty of multiplex PCR targeted amplification library construction. Therefore, even if the multiplex PCR method has a good library construction effect for the human genome, In the detection of multiplexed libraries of pathogenic microorganisms, the following problems still exist: 1) primers interfere with each other, forming a large number of dimers and reducing the performance of the kit; 2) amplified products are easily contaminated, resulting in false positives; 3) there are many operation steps and the library construction time is long; 4) non-specific amplification produces a large number of non-target amplicons, affecting subsequent sequencing.

Summary of the invention

According to the first aspect, in one embodiment, a cleavable circular primer is provided, comprising: a universal primer sequence, a target specific sequence, wherein the target specific sequence comprises at least one ribonucleotide.

According to the second aspect, in one embodiment, a kit is provided, comprising any cleavable circular primer of the first aspect.

According to the third aspect, in one embodiment, a PCR amplification method is provided, comprising:

A nucleic acid sample is provided, and the nucleic acid sample is subjected to one-step PCR amplification using any one of the kits of the second aspect to obtain an amplified product.

According to the fourth aspect, in one embodiment, there is provided a method for preparing the cleavable circular primer according to any one of the first aspects, comprising:

Providing a linear primer, removing the temporary blocking group at the 5' end of the linear primer, then modifying the phosphate group at the 5' end of the linear primer, and performing a cyclization reaction to obtain the cleavable circular primer;

The linear primer comprises a universal primer sequence and a target-specific sequence, wherein the target-specific sequence comprises at least one ribonucleotide, and the 5' end of the linear primer is modified with a temporary blocking group, and the 3' end is modified with a hydroxyl group.

Technical Effects

In one embodiment, the cleavable loop primers designed in the present disclosure effectively avoid the formation of dimers between primers.

In one embodiment, the highly specific shearing properties of ribonucleotides improve both the efficiency of the conversion from circular primers to linear primers and the efficiency of highly specific target amplification.

In one embodiment, the looped primer structure designed in the present disclosure further improves the stability of the primer and thus improves the stability of amplification.

In one embodiment, due to the high specificity and low dimer characteristics of the cleavable loop primer, the present disclosure significantly reduces the difficulty of multiple primer design, further improves product design and development efficiency, and reduces overall costs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic structural diagram of a loop primer according to an embodiment;

FIG2 is a flow chart of one-step multiplex amplification library construction according to an embodiment;

Fig. 3 is the agarose gel detection result in Example 4;

FIG4 is a schematic diagram of the structure of a single-stranded linear DNA primer according to an embodiment;

FIG5 is a flow chart of preparing a circular primer in one embodiment;

FIG6 is a schematic diagram of the state of a linear primer that does not participate in a cyclization reaction in an amplification system in one embodiment;

FIG7 is a schematic diagram showing the state of concatemeric DNA ring byproducts that may be produced by a cyclization reaction in an amplification system in one embodiment;

FIG8 is a schematic diagram showing the state of a single-stranded DNA copolymer byproduct that may be produced by a cyclization reaction in an amplification system in one embodiment;

FIG. 9 is a graph showing the electrophoresis results of circular primers after circularization of single-stranded linear primers.

Detailed ways

The present disclosure is further described in detail below through specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present disclosure to be better understood. However, those skilled in the art can easily recognize that some of the features can be omitted in different situations, or can be replaced by other materials or methods. In some cases, some operations related to the present disclosure are not shown or described in the specification, in order to avoid the core part of the present disclosure being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and they can fully understand the related operations based on the description in the specification and the general technical knowledge in the art.

In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence unless otherwise specified. Indicates that a certain order must be followed.

The serial numbers assigned to the components in this article, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning.

As used herein, "ribonucleotide" (ribotide) is composed of one molecule of phosphoric acid, one molecule of ribose (a five-carbon sugar), and one molecule of nitrogenous base. Ribonucleotides include adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, and uracil ribonucleotide.

As used herein, "deoxyribonucleotide" is a small molecule monomer of DNA (deoxyribonucleic acid). Each deoxyribonucleotide consists of three parts: a base, a deoxyribose, and a phosphate group. The nitrogenous base is bound to the 1' carbon atom of the deoxyribose, the phosphate group is bound to the 5' carbon atom of the deoxyribose, and the 2' carbon atom of the deoxyribose is connected to an H atom instead of -OH. Deoxyribonucleotides include adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymine deoxyribonucleotide.

As used herein, "one-step method" refers to adding multiple pairs of primers into the same reaction system to achieve multiplex PCR amplification, thereby achieving the detection of multiple target sequences.

As used herein, "complete universal primer" refers to a sequence that can be reverse complementary to a partial sequence in a primer connected to a sequencing adapter. In a primer connected to a sequencing adapter, the sequencing adapter is usually located at the 5' end, and the sequence that can be reverse complementary to the universal primer is usually located at the 3' end.

When deoxyribonucleotides are polymerized into DNA, the phosphate group will combine with the 3' carbon atom of the deoxyribose of another deoxyribonucleotide to form a phosphodiester bond through an esterification reaction. New nucleotides are usually added to the 3' carbon atom of the previous nucleotide, so the direction of DNA synthesis is from the 5' to the 3' end.

In view of the problems existing in the existing technology, there is an urgent need to develop a multiple targeted amplification library construction technology that can avoid contamination between samples during targeted amplification, reduce the difficulty of primer design, avoid the generation of primer dimers, and can be applied to the detection of pathogenic microorganisms.

According to the first aspect, in one embodiment, a cleavable circular primer is provided, comprising: a universal primer sequence, a target specific sequence (also called a targeting primer sequence), wherein the target specific sequence comprises at least one ribonucleotide (also called an RNA residue).

In one embodiment, the cleavable circular primer used in the present disclosure is a completely closed circular primer, which needs to remove RNA residues to open the circular shape and has good stability.

In one embodiment, the universal primer sequence is a deoxyribonucleotide sequence.

In one embodiment, in the target specific sequence, except for at least one ribonucleotide, the remaining nucleotides are all deoxyribonucleotides.

In one embodiment, the ribonucleotide sequence in the target specific sequence is a continuous or non-continuous sequence, preferably a continuous sequence.

In one embodiment, the target specific sequence comprises a ribonucleotide.

In one embodiment, the target specific sequence includes a deoxyribonucleotide located downstream of the at least one ribonucleotide, and the base sequence in the deoxyribonucleotide can be reverse complementary to the base sequence in the target sequence of the template nucleic acid molecule. The deoxyribonucleotide downstream of the ribonucleotide plays a protective role for the ribonucleotide, and is also called a protective sequence.

In one embodiment, the target specific sequence includes a deoxyribonucleotide located upstream of the at least one ribonucleotide and a deoxyribonucleotide located downstream of the at least one ribonucleotide. The deoxyribonucleotides upstream and downstream of the ribonucleotide can be at least one, preferably at least three, so that the ribonucleotide is efficiently removed in the subsequent steps.

In one embodiment, the base in the ribonucleotide is mismatched with the base in the target sequence of the template molecule, and the deoxyribonucleotides before and after the ribonucleotide are correctly paired with the base in the target sequence. The ribonucleotide can also be endonucleated, but the endonuclease efficiency is not as high as the correct reverse complementary pairing of the base sequence on the ribonucleotide with the base sequence in the target sequence of the template molecule.

In one embodiment, if there is a mismatch in the 1 to 3 positions before and after the ribonucleotide, the ribonucleotide cannot be internally cut; multiple ribonucleotides connected (the bases in the ribonucleotides are correctly paired with the bases in the target sequence) can also be cut together.

In one embodiment, ribonucleotides are spaced in the target-specific sequence and separated by deoxyribonucleotides in the target-specific sequence, and ribonucleotides can also be effectively removed.

In one embodiment, the universal primer sequence comprises a universal primer tail sequence located upstream of the target specific sequence and a universal primer front sequence located downstream of the target specific sequence.

In one embodiment, the 5' end of the universal primer rear sequence is connected in series with the 3' end of the universal primer front sequence to form a closed circular primer structure, that is, a completely closed circular primer.

In one embodiment, in the target-specific sequence, the base sequence in the deoxyribonucleotide is used for reverse complement pairing with the base sequence in the target sequence of the template nucleic acid molecule (ie, the nucleic acid molecule derived from the sample to be tested).

In one embodiment, the universal primer tail sequence is a sequence close to or located at the 3' end of the complete universal primer.

In one embodiment, the universal primer front sequence is a sequence close to or located at the 5' end of the complete universal primer.

In one embodiment, the target-specific sequence is used to specifically target a target sequence of a nucleic acid molecule, typically by binding to the target sequence through reverse complementary pairing.

In one embodiment, the template nucleic acid molecule is derived from a microorganism.

In one embodiment, the microorganism includes, but is not limited to, bacteria, fungi, protists, or viruses.

In one embodiment, the Tm value of the target specific sequence may be 50-65°C, preferably 56-60°C.

In one embodiment, the GC content of the target specific sequence may be 20-85%, preferably 30-70%.

In one embodiment, the target specific sequence comprises 10 to 30 nucleotides.

In one embodiment, the target specific sequence comprises ribonucleotides including, but not limited to, 1 to 20.

In one embodiment, the base sequence in at least one ribonucleotide is reverse complementary to or mismatched with the base sequence in the target sequence of the template nucleic acid molecule. In other words, the base in the ribonucleotide can be correctly paired with the target sequence or can be mismatched.

In one embodiment, in the target specific sequence, the base in the ribonucleotide may be at least one of A (adenine), U (uracil), C (cytosine), and G (guanine).

In one embodiment, the universal primer in the cleavable loop primer may specifically be a universal primer of a sequencing platform.

In one embodiment, as shown in FIG. 2 , the universal primer sequence in the cleavable loop primer can be reverse-complementarily paired with a portion of the sequence in the universal sequencing primer (a primer connected to a sequencing adapter). After the universal sequencing primer is added, the universal primer sequence in the cleavable loop primer is reverse-complementarily paired with at least a portion of the sequence in the universal sequencing primer to achieve PCR amplification and construct a complete library.

According to the second aspect, in one embodiment, a kit is provided, comprising any cleavable circular primer of the first aspect.

In one embodiment, the kit further comprises an enzyme.

In one embodiment, the enzyme includes ribonucleotide cleavage enzyme and DNA polymerase. Ribonuclease cleavage enzyme is also called endoribonuclease, which is used to cleave ribonucleotides in the circular primer.

In one embodiment, the ribonucleotide cleavage enzyme includes but is not limited to at least one of RNaseA, DpnII, RNaseH, RNaseH2, and RNaseH3.

In one embodiment, the DNA polymerase includes but is not limited to a thermostable DNA polymerase.

In one embodiment, the thermostable DNA polymerase includes but is not limited to at least one of Taq DNA polymerase and high-fidelity DNA polymerase.

In one embodiment, the kit further comprises a buffer.

In one embodiment, the buffer includes but is not limited to at least one of water, MgCl ₂ , dNTP, (NH ₄ ) ₂ SO ₄ , Triton, NaCl, Tris-HCl, KCl, BSA, glycerol, betaine, and PEG6000.

In one embodiment, in the kit, the reaction volume of the buffer is 10-100 μL; the water is double distilled water (ddH ₂ O), nuclease-free water or DEPC water; Tris-HCl is the pH buffer of the buffer, and the pH is 6.5-9.0; the concentration of MgCl ₂ is 0.5-8.0 mM; the concentration of dNTP is 100-500 mM each; the concentration range of NaCl and KCl is 10-150 mM; the volume percentage concentration of Triton is 0.001-1%; the concentration of BSA is 0.1-5 μg/μL; the concentration of glycerol is 1-10%; the concentration of betaine is 1-10 mM; the mass percentage concentration of PEG6000 is 1%-10%.

In one embodiment, the reaction volume of the buffer is 20-50 μL; the pH of Tris-HCl is 7.5-8.5; the concentration of MgCl ₂ is 1.0-5.0 mM; the concentration of dNTP is 150-250 mM each; the concentration of NaCl and KCl is 30-100 mM; the volume percentage concentration of Triton is 0.01-0.5%; the concentration of BSA is 0.4-0.8 μg/μL; the concentration of glycerol is 3-6%; the concentration of betaine is 2-5 mM; and the mass percentage concentration of PEG6000 is 1%-5%.

In one embodiment, the kit further comprises primers containing sequencing adapters, which are used to amplify library molecules that can be used for sequencing.

According to the third aspect, in one embodiment, a PCR amplification method is provided, comprising:

A nucleic acid sample is provided, and the nucleic acid sample is subjected to one-step PCR amplification using any one of the kits of the second aspect to obtain an amplified product.

In one embodiment, loop primers of multiple target sequences can be added to the reaction system to achieve amplification of multiple target sequences.

In one embodiment, the PCR amplification procedure includes:

Pre-denaturation: 93-98°C, 1-10 min;

Ribonucleotide shearing: 37-60°C, 1-10 min;

Cyclic amplification: 59-65°C, 1-5min.

In one embodiment, the nucleic acid sample includes but is not limited to a nucleic acid sample derived from a human or animal body. If the nucleic acid sample contains a target sequence, it will be detected. The target sequence can specifically be at least a partial nucleic acid sequence of a microorganism.

In one embodiment, the nucleic acid sample is derived from a body fluid sample and/or a tissue sample of a host.

In one embodiment, the method further includes detecting the amplification product.

In one embodiment, the detection includes but is not limited to gel electrophoresis analysis and/or sequencing analysis. Sequencing analysis includes but is not limited to second generation sequencing analysis.

According to the fourth aspect, in one embodiment, there is provided a method for preparing the cleavable circular primer according to any one of the first aspects, comprising the following steps:

A linear primer is provided, a temporary blocking group at the 5' end of the linear primer is removed, and then a cyclization reaction is performed after modifying the phosphate group at the 5' end of the linear primer to obtain a cleavable circular primer.

The linear primer comprises a universal primer sequence and a target-specific sequence, wherein the target-specific sequence comprises at least one ribonucleotide, the 5' end of the linear primer is modified with a temporary blocking group, and the 3' end is modified with a hydroxyl group. The linear primer can be used to synthesize any cleavable circular primer of the first aspect.

In one embodiment, the cleavable loop primer used in the present disclosure is a completely closed loop primer.

In one embodiment, the universal primer sequence is a deoxyribonucleotide sequence.

In one embodiment, in the target specific sequence, except for at least one ribonucleotide, the remaining nucleotides are all deoxyribonucleotides.

In one embodiment, the universal primer sequence comprises a universal primer tail sequence located upstream of the target specific sequence and a universal primer front sequence located downstream of the target specific sequence.

In one embodiment, the universal primer tail sequence refers to the sequence near or located at the 3' end of the complete universal primer.

In one embodiment, the universal primer front sequence refers to the sequence near or located at the 5' end of the complete universal primer.

In one embodiment, the temporary blocking group includes, but is not limited to, a hydroxyl group, a thiol group, or a phosphate group.

In one embodiment, the target specific sequence further comprises a deoxyribonucleotide located downstream of the at least one ribonucleotide, and the deoxyribonucleotide plays a protective role on the upstream ribonucleotide.

In one embodiment, the target specific sequence comprises a deoxyribonucleotide located upstream of the at least one ribonucleotide, and a deoxyribonucleotide located downstream of the at least one ribonucleotide.

In one embodiment, the number of deoxyribonucleotides upstream and downstream of the ribonucleotide may be at least one

In a preferred embodiment, the number of deoxyribonucleotides upstream and downstream of the ribonucleotide is at least two.

In a preferred embodiment, the number of deoxyribonucleotides upstream and downstream of the ribonucleotide is at least three, so that the ribonucleotide can be efficiently removed in the subsequent steps.

In one embodiment, the removal of the temporary blocking group at the 5' end of the linear primer, the modification of the phosphate group, and the cyclization reaction are completed in the same reaction system, without the need to separate into multiple systems for step-by-step reactions.

In one embodiment, a 5' phosphorylase is used to remove the temporary blocking group at the 5' end of the linear primer and to modify the phosphate group at the 5' end of the linear primer.

In one embodiment, the circularization reaction is performed using a single-stranded DNA ligase.

According to the fifth aspect, in one embodiment, a kit for preparing a cleavable circular primer is provided, the kit comprising a linear primer, the linear primer comprising a universal primer sequence, a target-specific sequence, the target-specific sequence comprising at least one ribonucleotide, the 5' end of the linear primer being modified with a temporary blocking group, and the 3' end being modified with a hydroxyl group. The linear primer can be used to synthesize the cleavable circular primer of any one of the first aspects.

In one embodiment, the kit further comprises an enzyme.

In one embodiment, the enzyme comprises at least one of 5' phosphorylase and single-stranded DNA ligase. The enzymes in the kit can all be purchased from the market.

In one embodiment, the kit further comprises ATP.

In one embodiment, the kit further comprises a buffer.

In one embodiment, the buffer includes but is not limited to Tris-HCl buffer.

In one embodiment, the kit further comprises other reagents required for the reaction, including but not limited to at least one of MgCl ₂ , DTT (dithiothreitol), Triton, polyethylene glycol, and the like.

In one embodiment, polyethylene glycol includes but is not limited to PEG8000.

In one embodiment, the present disclosure provides a supporting reagent for one-step multiplex PCR amplification, wherein the supporting reagent includes a cleavable circular primer.

In one embodiment, the supporting reagents also include an amplification reaction system.

In one embodiment, the amplification reaction system includes an enzyme and a buffer.

In one embodiment, the enzyme comprises a ribonucleotide cleavage enzyme and a DNA polymerase.

In one embodiment, the ribonucleotide cleavage enzyme is selected from any one or two of RNaseA, DpnII, RNaseH, RNaseH2, and RNaseH3.

In one embodiment, the DNA polymerase is Taq PCR polymerase or a high-fidelity PCR polymerase.

In one embodiment, the buffer comprises ddH ₂ O, MgCl ₂ , dNTP, (NH ₄ ) ₂ SO ₄ , Triton, NaCl, Tris-HCl, KCl, BSA, glycerol, betaine, and PEG6000.

In one embodiment, the reaction volume of the buffer is 10-100 μL; ddH ₂ O is double distilled water or nuclease-free water; Tris-HCl is the buffer pH buffer, and the pH is 6.5-9.0; the concentration of MgCl ₂ is 0.5-8.0 mM; the concentration of dNTP is 100-500 mM each; the concentration range of NaCl and KCl is 10-150 mM; the concentration of Triton is 0.001-1% by volume; the concentration of BSA is 0.1-5 μg/μL; the concentration of glycerol is 1-10%; the concentration of betaine is 1-10 mM; and the concentration of PEG6000 is 1%-10%.

In one embodiment, the present disclosure provides a one-step multiplex PCR amplification method, the multiplex PCR amplification method comprising:

(1) synthesizing a cleavable circular primer for one-step multiplex PCR amplification and preparing it into a solution;

(2) preparing a buffer solution and adding an enzyme mix to prepare an amplification reaction system;

(3) using a solution capable of cleaving the circular primer and an amplification reaction system, starting a multiplex PCR amplification procedure to perform a one-step multiplex PCR amplification;

(4) Analyze the amplification results.

In one embodiment, the PCR amplification procedure includes preliminary denaturation, cleavage of circular primers, and cyclic amplification.

In one embodiment, the result analysis includes gel electrophoresis analysis and/or sequencing analysis.

In one embodiment, the one-step multiplex PCR amplification method provided by the present disclosure comprises the following steps:

(1) preparing a target specific sequence containing ribonucleotides, connecting the rear end of a universal primer to the 5' end of the target specific sequence, and then connecting the front end of the universal primer to the 3' end of the target specific sequence to obtain a linear primer that can be used for one-step multiplex PCR amplification (as shown in FIG. 4 ), connecting the two ends of the linear primer to obtain a cleavable circular primer, and preparing a solution;

(2) using deionized water to prepare a buffer solution containing ddH ₂ O, MgCl ₂ , dNTP, (NH ₄ ) ₂ SO ₄ , Triton, NaCl, Tris-HCl, KCl, BSA, glycerol, betaine, and PEG6000, adding ribonucleotide cleavage enzyme and DNA polymerase to prepare an amplification reaction system;

(3) Performing PCR amplification using a solution capable of cleaving the circular primer and an amplification reaction system, wherein the PCR amplification procedure includes:

Pre-denaturation: 93-98°C, 1-10 min;

Ribonucleotide shearing: 37-60°C, 1-10 min;

Cyclic amplification: 59-65°C, 1-5 min;

(4) Perform gel electrophoresis analysis and/or sequencing analysis on the amplification results.

In one embodiment, in a cleavable circular primer, when at least part of the sequence before and after the ribonucleotide in the target specific sequence is reversely complementary paired with the template sequence, the ribonucleotide cleavage enzyme is activated to produce activity, and the ribonucleotide cleavage enzyme can specifically recognize the RNA residues in the primer, digest the RNA residues, and then convert the non-extendable circular primer into an extendable linear primer, and then the DNA polymerase extends and amplifies under the guidance of the linear primer to obtain the target product. The cleavable circular primer not only improves the specificity, but also achieves efficient amplification efficiency; the DNA protection sequence can prevent the RNA residues from being sheared by unnecessary external forces, and it is also a guarantee for the RNA residues to be efficiently sheared after being correctly paired with the template. While improving the efficiency of multiple PCR amplification, the generation of primer dimers is greatly reduced, and one-step amplification and library construction can be performed.

In one embodiment, in the target specific sequence, in the sequence before and after the ribonucleotide (i.e., RNA residue), the residue near the ribonucleotide The base sequence in at least one deoxyribonucleotide is reverse complementary to the template sequence, and the ribonucleotide can be removed by the corresponding cutting enzyme.

In a preferred embodiment, in the target specific sequence, in the sequence before and after the ribonucleotide (i.e., RNA residue), the base sequences in at least two deoxyribonucleotides close to the ribonucleotide are reverse complementary to the template sequence, and the ribonucleotide can be removed by the corresponding cutting enzyme.

In a preferred embodiment, in the target specific sequence, in the sequence before and after the ribonucleotide (i.e., RNA residue), the base sequence of at least three deoxyribonucleotides close to the ribonucleotide is reverse complementary to the template sequence, and the ribonucleotide can be removed more efficiently by the corresponding cutting enzyme.

In one embodiment, in the absence of a 100% correct pairing of a template, there is no linear primer sequence, which cannot hybridize and amplify normally, so the formation of primer dimers can be completely eliminated, and the occurrence of nonspecific amplification can be further reduced. Circular primers can also improve the stability of the primers themselves, so that the amplified products by amplification and sequencing adapters can be used for detection by second-generation sequencing technology, and the analysis efficiency is high. After the primers are cyclized, the amplification uniformity is good, and the requirements for mutual interference between primers are low, which greatly reduces the difficulty of primer design. Primers can also be added, subtracted or supplemented at any time according to target requirements, and the applicability and flexibility are stronger.

In one embodiment, the present disclosure provides a method for preparing a loop primer comprising the following steps:

Submit the targeting primer sequence to a primer synthesis company to synthesize single-stranded linear DNA primers;

Dissolve the synthesized primers in TE or nuclease-free water and dilute to a concentration of 100 μM;

Using 5' phosphorylase and single-stranded DNA ligase to react together, the dissolved and diluted primers are 5' phosphorylated and circularized;

The circularized products can be mixed into a primer pool for multiplex targeted amplification reactions without purification.

In one embodiment, the present disclosure provides a method for preparing a circular primer using a single-stranded linear DNA primer.

In one embodiment, as shown in Figures 1, 2, and 4, the universal primer is divided into two sections, specifically the universal primer front section and the universal primer back section, the universal primer back section refers to the partial sequence near the 3' end of the universal primer complete sequence, and the front section refers to the sequence near the 5' end of the universal primer complete sequence. The single-stranded linear DNA primer includes a forward unimolecular primer and a reverse unimolecular primer, which sequentially contain the universal primer back section, the target-specific sequence, and the universal primer front section from the 5'-3' direction, and the target-specific sequence contains at least one ribonucleotide. In the target-specific sequence, except for ribonucleotides, the rest are deoxyribonucleotides, and the base sequence in at least part of the deoxyribonucleotides can be reversely complementary paired with the base sequence in the target sequence of the template nucleic acid molecule. There is at least one deoxyribonucleotide upstream and downstream of the ribonucleotide. As shown in Figure 5, this structure effectively avoids the negative impact of the above-mentioned by-products on the amplification system when the single-stranded linear DNA primer fails to form a ring or single-stranded DNA copolymers and concatemer DNA rings appear during the connection process.

As shown in Figure 2, the circular primer is hybridized and paired with the template, the single-molecule tag enzyme digests the RNA residue, the circular primer is uncirculated, the first part is free in the reaction system, 3-OH is exposed, and the DNA polymerase is extended and amplified. Then, the primer containing the sequencing adapter is used to amplify the targeted amplification product with a universal primer to construct a complete library.

The present invention divides the complete universal primer into two parts, which are connected to the two ends of the linear primer respectively. Therefore, when the linear primer fails to form a loop, it will not hybridize with the subsequent library amplification primer and amplify incorrectly, thereby forming a protection mechanism. After the linear primer forms a loop, the universal primer sequences of the two parts are recombined into a complete sequence, and when the circular primer is cut from the ribonucleotide, the universal primer sequence remains intact and hybridizes with the subsequent library amplification primer, thereby correctly amplifying the target library.

In one embodiment, the rear section and the front section of the universal primer are both universal sequences of non-homologous sequences.

In one embodiment, when there is only one or a continuous ribonucleotide in the target specific sequence of the cleavable loop primer, the sequence located upstream of the ribonucleotide in the target specific sequence can be called the target specific sequence front segment, and the sequence located downstream of the ribonucleotide in the target specific sequence can be called the target specific sequence rear segment, and the target specific sequence front segment and rear segment sequences are a specific sequence designed using bioinformatics methods for the target amplification sequence. The target specific sequence front segment refers to the sequence located upstream of the ribonucleotide (i.e., the 5' end), and the target specific sequence rear segment refers to the sequence located downstream of the ribonucleotide (i.e., the 3' end).

In one embodiment, the target specific sequence of the cleavable loop primer may contain multiple spaced single ribonucleotides or continuous ribonucleotide sequences, and the base sequence of the deoxyribonucleotides other than the ribonucleotides in the target specific sequence may be reverse complementary to the base sequence at the corresponding position in the target sequence of the template nucleic acid molecule.

In one embodiment, the base sequences in the deoxyribonucleotides and ribonucleotides in the target specific sequence of the cleavable loop primer can be reverse complementary paired with the base sequences at corresponding positions in the target sequence of the template nucleic acid molecule.

In one embodiment, the number of bases in the rear section of the universal primer is 0 to 30; the Tm value of the front section of the target specific sequence is 50 to 65°C, and the GC The content is 20-85%, the number of bases is 10-35; the number of RNA residues is 1-20; the type of RNA residues is any one or more random combinations of A, T, C, G, and U; the number of bases in the latter part of the target specific sequence is 1-15; the number of bases in the front part of the universal primer is 10-40.

In one embodiment, the front and back ends of the universal primer are both sequencing primer sequences; the number of bases in the back end of the universal primer is 0 to 20; the Tm value of the target specific sequence is 56 to 60°C, and the GC content is 30 to 70%; the number of RNA residues is 1 to 10; the number of bases in the back end of the target specific sequence is 4 to 10; and the number of bases in the front end of the universal primer is 20 to 40.

In one embodiment, the present disclosure provides a method for circularizing a single-stranded linear DNA primer, wherein the reagents used include a circularizing enzyme preparation and a reaction buffer.

In one embodiment, the enzyme preparation comprises two enzymes: 5' phosphorylase and single-stranded DNA ligase; the 5' phosphorylase recognizes the 5' hydroxyl group in the single-stranded linear DNA primer and phosphorylates the 5' hydroxyl group to a 5' phosphate group in the environment of reaction buffer and ATP; the single-stranded DNA ligase catalyzes the intramolecular connection (i.e., circularization) of the ssDNA template with a 5' phosphate group and a 3' hydroxyl group, so that the 5' phosphorylated single-stranded linear DNA primer can normally form a phosphodiester bond with the 3' hydroxyl group, thereby connecting the single-molecule primer into a ring.

In one embodiment, the 5' phosphorylase is selected from any one or both of T4PNK and PNPase.

In one embodiment, the single-stranded DNA ligase is selected from at least one or two of ssDNA CircLigase and T4 RNA Ligase.

In one embodiment, the reaction buffer comprises water, MgCl ₂ , DTT, Triton, Tris-HCl, PEG8000, and ATP.

In one embodiment, the reaction volume of the reaction buffer is 10-100 μL; water is double distilled water (ddH ₂ O) or nuclease-free water; Tris-HCl is the buffer, and the pH is 6.5-9.0; the concentration of DTT is 2-15.0 mM; the concentration of MgCl ₂ is 2-20.0 mM; the concentration of Triton is 0.01-0.5% by volume; the mass percentage concentration of PEG8000 is 1%-10%; and the concentration of ATP is 0.5-5.0 mM.

In one embodiment, the reaction volume of the reaction buffer is 20-50 μL; the pH of Tris-HCl is 7.5-8.5; the concentration of DTT is 3-8.0 mM; the concentration of MgCl ₂ is 5.0-15.0 mM; the concentration of Triton is 0.05-0.2% by volume; the concentration of PEG8000 is 1%-5%; and the concentration of ATP is 0.5-2.0 mM.

In one embodiment, the circular primer preparation method disclosed in the present invention can simultaneously perform 5' phosphorylation and single-stranded DNA circularization. Due to the special enzymatic activity of single-stranded DNA ligase, it connects ssDNA in the absence of complementary sequences, and does not produce detectable single-stranded DNA copolymers or concatemer DNA rings under standard reaction conditions. With the use of special enzyme preparations, a unique reaction buffer can be achieved in which both 5' phosphorylation and the activity of single-stranded DNA ligase can be fully exerted, thereby achieving the purpose of one-step circularization of single-stranded primers.

In one embodiment, after the 5' phosphorylation and cyclization reactions are completed, there is no need to purify the reaction products or digest the linear DNA. As shown in FIG6, FIG7, and FIG8, due to the special structure of the synthesized single-stranded linear DNA primer, even if there is single-stranded linear DNA that does not participate in the reaction, or single-stranded DNA copolymers and concatemer DNA rings appear, it has almost no effect on the construction of multiple targeted libraries.

As shown in Figure 6, the uncircularized primers are hybridized and paired with the template, the single-molecule tag enzyme digests the RNA residues, the uncircularized primers are broken, the universal primer front segment is free in the reaction system, 3-OH is exposed, and the DNA polymerase extends and amplifies. When the universal primer amplification is performed on the targeted amplification product, the amplification product cannot be amplified because it lacks the universal primer front segment.

As shown in Figure 7, concatemer DNA rings can be cut by single-molecule tag enzymes, circular primers are unwound, 3'-OH is exposed, and DNA polymerases are extended for amplification. However, the amplification efficiency of long primers is low, and the more concatemers there are, the lower the amplification efficiency. The amplification products of concatemer DNA rings can be amplified by universal primers. The first cycle has the most amplification combinations. Since the amplification efficiency of short fragments is higher than that of long fragments, as the number of cycles increases, only the shortest library sequence is slowly formed.

As shown in Figure 8, the single-stranded linear DNA copolymer can be cut by the single-molecule tag enzyme, bind to one end of the template, expose the 3'-OH, and extend the DNA polymerase for amplification. However, the amplification efficiency of long primers is low, and the more concatemers there are, the lower the amplification efficiency. When amplifying with universal primers, two situations will occur:

Case 1: The amplification product of the single-stranded linear DNA copolymer can be amplified by universal primers. The first cycle has the most amplification combinations. Since the amplification efficiency of short fragments is higher than that of long fragments, as the number of cycles increases, only the shortest library sequence is gradually formed.

Case 2: Only one end of the amplification product of the single-stranded linear DNA copolymer can be amplified by the universal primer and cannot be amplified.

In the prior art, the primers used for amplification are all linear nucleic acid primers, and there has been no precedent reported for amplification using circular primers, nor has there been any method for preparing circular primers.

In one embodiment, the circular multiple primers disclosed herein contain the enzyme preparation and buffer of the above-mentioned primer pool, and can complete the construction of single-stranded linear DNA molecules into single-stranded circular DNA molecules through only one round of enzyme reaction, without the need for complex processes such as chemical pathways and modification of the chemical structure of nucleic acid molecules.

In one embodiment, the single-stranded linear DNA molecular structure disclosed in the present invention can effectively avoid non-specific amplification and primer dimers generated in the amplification system by a small number of single-stranded linear primer molecules, single-stranded DNA copolymers or concatemer DNA loops that fail to form single-stranded DNA loops due to 5' phosphorylation.

Example 1

This embodiment provides a method for preparing a cleavable circular primer, which is as follows:

1. Single-stranded linear DNA primer design: Design amplification primers that meet the requirements for the target regions of several pathogens; arrange the universal primer rear section, target specific sequence front section, RNA residues, target specific sequence rear section, and universal primer front section information according to the requirements of single-stranded linear DNA primers, and send them to the primer synthesis company for primer synthesis.

2. Preparation of system 2× cyclization buffer: prepare chemical reagents such as ddH ₂ O, MgCl ₂ , DTT, Triton, Tris-HCl, PEG8000, and ATP according to the preferred concentrations.

3. Prepare the following system for primer circularization. Single-stranded linear primers can be mixed to form a multiple targeted primer pool and then circularized. Single primers can also be circularized.

Table 1

The cyclase preparation was prepared from T4 Polynucleotide Kinase with catalog number M0201V purchased from NEB and CircLigase II ssDNA Ligase with catalog number CL9021K purchased from Lucigen at an activity unit ratio of 1:100.

4. Carry out the reaction according to the following procedure, with the lid heated at 65°C.

Table 2

5. After the cyclization reaction is completed, there is no need to purify or digest the linear DNA. It can be directly diluted and mixed into a multiple targeted primer pool for use.

6. The electrophoresis result of the circular primer after circularization of the single-stranded linear primer is shown in FIG9 .

The description in FIG. 9 is as follows: M: marker; 1: synthetic linear primer; 2: cleavable circular primer prepared by the present disclosure.

It can be seen that circular primers were basically formed, and no bands of single-stranded DNA copolymers or concatemer DNA rings were seen, indicating that the content of unexpected products was low.

Example 2

This example provides a set of cleavable circular primers for one-step multiplex PCR amplification, which can be used in the combined detection of Epstein-Barr virus, Candida albicans, Babesia, and Aspergillus flavus. The preparation method of the circular primers is as described in Example 1.

The sequences of the cleavable circular primers for detecting Epstein-Barr virus are shown in SEQ ID No. 1-2;

The sequences of the cleavable circular primers for detecting Candida albicans are shown in SEQ ID No. 3 to 4;

The sequences of the cleavable circular primers for detecting Babesia are shown in SEQ ID No. 5-6;

The sequences of the cleavable circular primers for detecting Aspergillus flavus are shown in SEQ ID No.7~8.

table 3

In Table 3, the underlined single straight line indicates the rear sequence of the universal primer, the underlined double straight line indicates the target specific sequence, the lowercase letters indicate the bases in the ribonucleotide (i.e., RNA bases), the underlined bold straight line indicates the DNA protection sequence, and the underlined wavy line indicates the front sequence of the universal primer. The 5' end of the rear sequence of the universal primer is connected to the 3' end of the front sequence of the universal primer to form a closed loop structure.

Example 3

This embodiment provides a set of cleavable circular primers for one-step multiplex PCR amplification, and the cleavable circular primers can be used in the joint detection of multiple pathogenic microorganisms.

The sequence of the aforementioned cleavable loop primer is shown in Table 4. Specifically,

The sequences of the cleavable circular primers for detecting Aspergillus niger are shown in SEQ ID No. 9 to 14;

The sequences of the cleavable circular primers for detecting Cryptococcus laurentii are shown in SEQ ID No. 15 to 20;

The sequences of the cleavable circular primers for detecting Chikungunya virus are shown in SEQ ID No. 21 to 26;

The sequences of the cleavable circular primers for detecting Enterobacter cloacae are shown in SEQ ID No. 27 to 32;

The sequences of the cleavable circular primers for detecting desulfurizing bacteria are shown in SEQ ID No. 33 to 38;

The sequences of the cleavable circular primers for detecting Mycobacterium abscessus are shown in SEQ ID No. 39 to 44;

The sequences of the cleavable circular primers for detecting Candida albicans are shown in SEQ ID No. 45 to 50;

The sequences of the cleavable circular primers for detecting Bacteroides fragilis are shown in SEQ ID No. 51 to 56;

The sequences of the cleavable circular primers for detecting Kingella denitrificans are shown in SEQ ID No. 57 to 62;

The sequences of the cleavable circular primers for detecting Cryptococcus neoformans are shown in SEQ ID No. 63 to 68;

The sequences of the cleavable circular primers for detecting Neisseria meningitidis are shown in SEQ ID No. 69 to 74;

The sequences of the cleavable circular primers for detecting Streptococcus salivarius are shown in SEQ ID No. 75 to 80;

The sequences of the cleavable circular primers for detecting the new bunyavirus are shown in SEQ ID No. 81 to 86;

The sequences of the cleavable circular primers for detecting Aspergillus pyrogenes are shown in SEQ ID No. 87 to 92;

The sequences of the cleavable circular primers for detecting Lactobacillus rhamnosus are shown in SEQ ID No. 93 to 98;

The sequences of the cleavable circular primers for detecting Aspergillus nidulans are shown in SEQ ID No.99～104.

Table 4

In Table 4, the underlined single straight line indicates the rear sequence of the universal primer, the underlined double straight line indicates the target specific sequence, the lowercase letters indicate the bases in the ribonucleotide (i.e., RNA bases), the underlined bold straight line indicates the DNA protection sequence, and the underlined wavy line indicates the front sequence of the universal primer. The 5' end of the rear sequence of the universal primer is connected to the 3' end of the front sequence of the universal primer to form a closed loop structure.

Example 4

The present embodiment provides a supporting reagent for one-step multiplex PCR amplification, the supporting reagent comprising the cleavable circular primer for one-step multiplex PCR amplification in Embodiments 2 and 3, an enzyme mixture and a buffer, the enzyme mixture comprising a thermostable RNaseH2 enzyme with the item number C5051 purchased from Xinhai Gene, a Taq DNA polymerase with the item number MD001 produced by Feipeng Bio, and a Q5 high-fidelity DNA polymerase with the item number M0491 purchased from NEB, the mixture is prepared according to the activity unit ratio of thermostable RNaseH2 enzyme: Taq DNA polymerase: Q5 high-fidelity DNA polymerase = 1:10:2.5; the buffer is 2×buffer, comprising ddH ₂ O, MgCl ₂ , dNTP, (NH ₄ ) ₂ SO ₄ , Triton, NaCl, Tris-HCl, KCl, BSA, glycerol, betaine, PEG6000, wherein the final concentration of MgCl ₂ is 8mM, the final concentration of dNTPs is 300mM, the final concentration of (NH ₄ ) ₂ SO ₄ is 15mM, the mass fraction of Triton is 0.1%, the final concentration of NaCl is 15mM, the final concentration of Tris-HCl is 20mM, the final concentration of KCl is 80mM, the mass volume ratio of PEG6000 is 3%, and the total concentration of betaine is 1.5M.

The supporting reagents provided by the present invention for one-step multiplex PCR amplification have good specificity and can significantly reduce the generation of primer dimers. Taq DNA polymerase can ensure high amplification efficiency, and Q5 high-fidelity enzyme can ensure that no mismatches and mutations are introduced during the amplification process and avoid the occurrence of nonspecific amplification. The optimized amplification reaction system further improves the specificity of the supporting reagents.

Example 5

This example uses the supporting reagents for one-step multiplex PCR amplification prepared in Example 4 to detect artificially synthesized template samples containing target sequences of Epstein-Barr virus, Candida albicans, Babesia, and Aspergillus flavus. The specific process includes PCR amplification and analysis.

The PCR amplification system is as follows:

table 5

The primers containing sequencing adapters include 5' end sequencing universal primers and 3' end sequencing universal primers, as follows:

5' end sequencing universal primer:

3' end sequencing universal primer:

In the universal sequencing primer, the sequence marked with an underline and italics is the sample tag sequence. The sequence marked with a wavy underline can be reverse-complemented with the partial sequence of the cleavable loop primer. The sequence not marked with an underline is the sequencing adapter.

The one-step multiplex PCR amplification procedure is as follows:

Table 6

Deionized water was used instead of sample template for amplification as a negative control group. Non-circular primers (linear primers, complete universal primers and target-specific sequences from 5' to 3' ends) were used as conditional control groups. The amplification results by agarose gel electrophoresis are shown in Figure 3.

As can be seen from Figure 3 (using all primers in Table 3), the bands amplified using the cleavable circular primers are brighter and have no mixed bands, indicating that the amplification efficiency is high and no primer dimers and non-specific amplification are produced; the negative control has no bands, proving that the amplification system is free of contamination; while the brightness of the amplified bands in the conditional control group using linear primers is darker and there are many mixed bands, indicating that its amplification efficiency is low and primer dimers and non-specific amplification are produced. This shows that the cleavable circular primers disclosed in the present invention have better performance.

Example 6

In this example, the supporting reagents for one-step multiplex PCR amplification constructed in Example 4 (using cleavable circular primers) are used, and the cleavable circular primers in Examples 2 and 3 are combined into a primer pool, 10 clinical blood samples with clinically confirmed reports and artificially synthesized template samples containing Epstein-Barr virus, Candida albicans, Babesia, and Aspergillus flavus target sequences are obtained, and the metagenomics (mNGS) and the method of the present disclosure are used for detection, respectively, to illustrate that the present disclosure has good scalability and strong adaptability, as well as the relative Compared with mNGS, it has a higher positive detection rate and has broad application prospects. The specific process includes one-step multiplex PCR amplification library construction and high-throughput sequencing analysis.

The system and procedure for one-step multiplex PCR amplification and library construction were the same as those in Example 5; mNGS was performed according to the instructions of the finished kit.

High-throughput sequencing analysis includes the following steps:

(1) Purify the amplified product using 0.8× purification magnetic beads;

(2) The purified amplified libraries were mixed and pooled at an equimolar ratio to obtain a library concentration of 1.9 pmol/μL;

(3) The resulting library was sequenced using the Illumina sequencing platform;

(4) Filter the raw data and remove low-quality data;

(5) Split the filtered data into sample data according to different index labels;

(6) Compare and analyze the sequencing sequences obtained by splitting with the constructed pathogenic microorganism database to identify the pathogenic bacteria.

The test results are shown in Table 7.

Table 7

It can be seen that the test results disclosed herein are 100% consistent with the clinical diagnosis results, and the positive detection rate is 30% higher than that of the mNGS test results, confirming that the cleavable circular primer disclosed herein has high accuracy and positive rate when used for pathogenic microorganism detection.

Comparative Example 1

This comparative example uses the same target sequence in Examples 2 and 3 to synthesize common non-circular structure primers, i.e., linear primers, which are then combined into a primer pool, and the same 10 clinical blood samples as in Example 6 and artificially synthesized template samples containing Epstein-Barr virus, Candida albicans, Babesia, and Aspergillus flavus target sequences are used for detection, and the detection results are compared with the results of the present disclosure to illustrate that the present disclosure has strong specificity and sensitivity and has great advantages. The specific process includes multiplex PCR amplification library construction and high-throughput sequencing analysis.

From the 5' end to the 3' end, there are a complete universal primer and a target specific sequence, the target specific sequence contains a ribonucleotide, and the 3' end of the entire sequence is modified with a blocking group, which is specifically a C3spacer modification group.

For example, the linear primer corresponding to the circular primer shown in SEQ ID No. 1 is as follows:

This comparative example designed linear primers corresponding to SEQ ID No. 1 to 104.

The system and procedure for multiplex PCR amplification library construction are as follows:

(1) The first round of multiplex PCR amplification system was prepared using the following system, wherein the enzyme mixture and buffer were the same as those in Example 4:

Table 8

(2) Use the following amplification reaction procedure:

Table 9

(3) Purify the amplified product using 0.8× purification magnetic beads;

(4) Use the following system to prepare the second round of multiplex PCR amplification system;

Table 10

(5) Use the following amplification reaction procedure:

Table 11

The high-throughput sequencing analysis steps are the same as those in Example 6;

The test results are shown in the following table.

Table 12

It can be seen that compared with linear primers, the number of reads of each microbial detection target in the present disclosure far exceeds that of ordinary linear primers under the same data volume, and the effective data volume directly affects the accuracy of the results. In summary, compared with traditional linear primers, the present disclosure has a significant performance improvement.

In one embodiment, the cleavable circular primers designed by the present disclosure avoid the formation of dimers between primers; the highly specific cleavage characteristics of RNA residues not only improve the efficiency of the conversion from circular primers to linear primers, but also improve the efficiency of highly specific target amplification; the circular primer structure further improves the stability of the primer and thus the stability of the amplification. The sequencing adapter allows the amplified product to be directly analyzed using the second-generation sequencing technology, and the analysis results are accurate. Due to the high specificity and low dimer characteristics of the cleavable circular primer, the difficulty of multiple primer design is significantly reduced, further improving the efficiency of product design and development and reducing overall costs.

In one embodiment, the cleavable circular primer disclosed in the present invention can complete the construction of library molecules through only one round of PCR amplification, significantly shortening the library construction time, avoiding contamination caused by too many operation steps, and reducing the cost of library construction.

In one embodiment, in the library construction method disclosed herein, the target region to be detected can be increased or decreased without affecting the design of other targets in the system. Therefore, the library construction method disclosed herein has good scalability, and the target region can be increased based on the target panel according to demand.

In one embodiment, the supporting reagents provided by the present disclosure for use in a one-step multiplex PCR amplification method improve the adaptability of the reaction system to cleavable circular primers by optimizing the amplification reaction system, resulting in less nonspecific amplification and primer dimers, higher amplification efficiency, and more accurate results; the amplification results have excellent uniformity, can maintain the relative proportion of the template target, and the results are closer to the actual situation; it is easy to use, easy to operate, highly applicable, and has broad application prospects.

The above specific examples are used to illustrate the present disclosure, which is only used to help understand the present disclosure and is not intended to limit the present disclosure. For those skilled in the art of the present disclosure, according to the idea of the present disclosure, some simple deductions, deformations or substitutions can be made.

Although the present disclosure has been illustrated and described with particular embodiments, it will be appreciated that many other changes and modifications may be made without departing from the spirit and scope of the present disclosure. Therefore, it is intended to include in the appended claims all such changes and modifications that fall within the scope of the present disclosure.

Industrial Applicability

The present disclosure provides a cleavable circular primer and a kit and an amplification method thereof. The amplification method is suitable for high-specific amplification of a target; the circular primer structure further improves the stability of the primer and thus improves the stability of amplification, and has broad application prospects and high market value.

Claims (10)

1. A cleavable circular primer, wherein the cleavable circular primer comprises: a universal primer sequence and a target specific sequence, and the target specific sequence comprises at least one ribonucleotide.
2. The cleavable circular primer according to claim 1, wherein the universal primer sequence is a deoxyribonucleotide sequence;

   Preferably, in the target specific sequence, except for the at least one ribonucleotide, the remaining nucleotides are deoxyribonucleotides;

   Preferably, the cleavable loop primer is a completely closed loop primer.
3. The cleavable circular primer according to claim 1, wherein the target specific sequence comprises a deoxyribonucleotide located downstream of the at least one ribonucleotide, and the base sequence in the deoxyribonucleotide can be reverse complementary to the base sequence in the target sequence of the template nucleic acid molecule;

   Preferably, the target specific sequence comprises a deoxyribonucleotide located upstream of the at least one ribonucleotide, and a deoxyribonucleotide located downstream of the at least one ribonucleotide;

   Preferably, the universal primer sequence comprises a universal primer rear sequence located upstream of the target specific sequence and a universal primer front sequence located downstream of the target specific sequence;

   Preferably, the 5' end of the universal primer rear sequence is connected in series with the 3' end of the universal primer front sequence;

   Preferably, in the target-specific sequence, the base sequence in the deoxyribonucleotide is used for reverse complementary pairing with the base sequence in the target sequence of the template nucleic acid molecule;

   Preferably, the template nucleic acid molecule is derived from a microorganism;

   Preferably, the target specific sequence contains 10 to 30 nucleotides;

   Preferably, the target specific sequence comprises 1 to 20 ribonucleotides.
4. The cleavable circular primer according to claim 1, wherein the base sequence in the at least one ribonucleotide is reverse-complementarily paired or mismatched with the base sequence in the target sequence of the template nucleic acid molecule.
5. A kit, wherein the kit comprises the cleavable circular primer according to any one of claims 1 to 4.
6. The kit according to claim 5, wherein the kit further comprises an enzyme;

   Preferably, the enzyme comprises ribonucleotide cleavage enzyme and DNA polymerase;

   Preferably, the ribonucleotide cleavage enzyme comprises at least one of RNaseA, DpnII, RNaseH, RNaseH2, and RNaseH3;

   Preferably, the DNA polymerase comprises a thermostable DNA polymerase;

   Preferably, the thermostable DNA polymerase comprises at least one of Taq DNA polymerase and high-fidelity DNA polymerase;

   Preferably, the kit further comprises a buffer.
7. A PCR amplification method, comprising:

   A nucleic acid sample is provided, and PCR amplification is performed on the nucleic acid sample using the kit according to any one of claims 5 to 6 to obtain an amplification product.
8. The PCR amplification method according to claim 7, wherein the PCR amplification procedure comprises:

   Pre-denaturation: 93-98°C, 1-10 min;

   Ribonucleotide shearing: 37-60°C, 1-10 min;

   Cyclic amplification: 59-65°C, 1-5 min;

   Preferably, the nucleic acid sample comprises a nucleic acid sample derived from a human or animal body;

   Preferably, the nucleic acid sample is derived from a body fluid sample and/or a tissue sample;

   Preferably, the method further comprises detecting the amplification product;

   Preferably, the detection comprises gel electrophoresis analysis and/or sequencing analysis.
9. The method for preparing a cleavable circular primer according to any one of claims 1 to 4, wherein the preparation method comprises the following steps:

   Providing a linear primer, removing the temporary blocking group at the 5' end of the linear primer, and then performing a cyclization reaction after modifying the phosphate group at the 5' end of the linear primer, thereby obtaining the cleavable circular primer;

   The linear primer comprises a universal primer sequence and a target-specific sequence, wherein the target-specific sequence comprises at least one ribonucleotide, and the 5' end of the linear primer is modified with a temporary blocking group, and the 3' end is modified with a hydroxyl group.
10. The preparation method according to claim 9, wherein the universal primer sequence comprises a universal primer rear sequence located upstream of the target specific sequence and a universal primer front sequence located downstream of the target specific sequence;

    Preferably, in the target specific sequence, except for the at least one ribonucleotide, the remaining nucleotides are deoxyribonucleotides;

    Preferably, the universal primer sequence is a deoxyribonucleotide sequence;

    Preferably, in the target specific sequence, except for the at least one ribonucleotide, the remaining nucleotides are deoxyribonucleotides;

    Preferably, the universal primer sequence comprises a universal primer rear sequence located upstream of the target specific sequence and a universal primer front sequence located downstream of the target specific sequence;

    Preferably, the temporary blocking group comprises a hydroxyl group, a thiol group or a phosphate group;

    Preferably, the target-specific sequence further comprises a deoxyribonucleotide located downstream of the at least one ribonucleotide;

    Preferably, the removal of the temporary blocking group at the 5' end of the linear primer, the modification of the phosphate group, and the cyclization reaction are completed in the same reaction system;

    Using 5' phosphorylase to remove the temporary blocking group at the 5' end of the linear primer, and modifying the phosphate group at the 5' end of the linear primer;

    Preferably, the circularization reaction is carried out using a single-stranded DNA ligase.