CN114250273A

CN114250273A - Composition for nucleic acid detection

Info

Publication number: CN114250273A
Application number: CN202011006675.9A
Authority: CN
Inventors: 赵雨航; 葛志琪; 何辉煌
Original assignee: Maccura Biotechnology Co ltd
Current assignee: Maccura Biotechnology Co ltd
Priority date: 2020-09-23
Filing date: 2020-09-23
Publication date: 2022-03-29
Anticipated expiration: 2040-09-23
Also published as: CN114250273B

Abstract

The invention discloses a high-sensitivity and high-specificity composition for nucleic acid detection and application of the composition in a detection kit. The composition comprises: a first upstream primer F1, a second upstream primer F2, a probe P, a downstream primer R and an oligonucleotide F1C; the first upstream primer consists of the following two parts from the 5 'end to the 3' end in sequence: (1) a mismatch area: no complementary pairing with the target nucleic acid sequence; the sequence of the mismatch region includes, from the 5 'end to the 3' end, the same sequence as the second forward primer, and the same sequence as the probe; (2) a matching area: complementary pairing occurs with the target nucleic acid sequence. Compared with the prior art, the primer probe composition provided by the invention has the remarkable advantages of high sensitivity, good specificity, low cost and the like, is suitable for nucleic acid detection of various sample types, and has extremely high application value.

Description

Composition for nucleic acid detection

Technical Field

The invention relates to the field of molecular biology, in particular to a primer probe composition for improving the sensitivity and specificity of nucleic acid detection and application thereof in PCR reaction.

Background

The Polymerase Chain Reaction (PCR) is a molecular biology technique for the enzymatic replication of DNA without using living organisms. PCR is commonly used in medical and biological research laboratories to undertake a variety of tasks, such as gene cloning, phenotypic identification of laboratory animals, transcriptome studies, detection of genetic diseases, identification of gene fingerprints, diagnosis of infectious diseases, paternity testing, and the like. Due to its incomparable replication and precision capabilities, PCR is considered by molecular biologists to be the first method of nucleic acid detection. In the later 90 s of the last century, Real Time Quantitative PCR (qPCR) technology and related products, which were introduced by ABI of America, developed PCR into a nucleic acid sequence analysis technology with high sensitivity, high specificity and accurate quantification.

The digital PCR (digital PCR) technique is an absolute nucleic acid molecule quantification technique, which uses the principle of limiting dilution to distribute a fluorescent quantitative PCR reaction system into thousands of individual nanoliter microreactors, so that each microreactor may or may not contain 1 or more copies of a target nucleic acid molecule (DNA target), and then simultaneously perform single-molecule template PCR amplification. Different from the method of acquiring fluorescence during each amplification cycle of the fluorescent quantitative PCR, the digital PCR independently acquires the fluorescence signal of each reaction unit after the amplification is finished, and finally obtains the original copy number or concentration of the target molecule according to the principle of Poisson distribution and the proportion of positive/negative reaction units.

Compared with fluorescent quantitative PCR, the digital PCR can carry out accurate absolute quantitative detection without depending on Ct value and a standard curve, and has the advantages of high sensitivity and high accuracy. Because the digital PCR only judges the 'existence/nonexistence' of two amplification states during result judgment, the intersection point of a fluorescence signal and a set threshold line does not need to be detected, and the method does not depend on the identification of a Ct value completely, so that the influence of the amplification efficiency on the digital PCR reaction and the result judgment is greatly reduced, and the tolerance capability on PCR reaction inhibitors is greatly improved. In addition, the process of allocating the reaction system in the digital PCR experiment can greatly reduce the concentration of the background sequence having competition effect with the target sequence locally. Therefore, digital PCR represents a significant advantage over traditional fluorescent quantitative PCR when quantification and detection of low copy number differential nucleic acid molecules with high sensitivity is required due to its higher sensitivity and accuracy. Especially, rare mutations are detected in complex backgrounds, and are often found in tumor liquid biopsy, noninvasive prenatal detection, organ transplantation monitoring, accurate quantification of viral load, component detection of transgenic crops and the like, for example, rare mutation markers are detected in peripheral blood of tumor patients, or gene expression difference research and the like.

The existing nucleic acid detecting reagent adopts TaqMan probe method, primer specificity distinguishing method and wild blocking type amplification method. The method comprises the steps of firstly, generally adopting competitive probes respectively aiming at a mutant type and a wild type, designing two probes to be competitive probes similar to point mutation detection, or designing a specific probe on a gene wild type template, designing a general probe capable of indicating the wild type template and the mutant type template at the same time in other conserved regions of a gene, and quantifying the concentration of the mutant type template by using the concentration difference measured by the two probes. The primer specificity distinguishing method is to design different mutant primers to distinguish different mutation types specifically, design a probe and a downstream primer in a downstream conserved region, and design a universal probe capable of indicating wild type and mutant template simultaneously and upstream and downstream primers in other conserved regions of the gene. ③ the wild-type amplification method is usually to adopt Peptide Nucleic Acid (PNA), Locked Nucleic Acid (LNA) and other expensive modifications to block the amplification of the wild-type template, so as not to generate false positive signals, but when the difference between some wild-type sequences and mutant sequences is too small or the mutant region is a wild-type repetitive sequence, the added inhibitor can not distinguish the two sequences and influence the mutant signal detection.

Therefore, a primer probe design method with high sensitivity, good specificity and low cost is needed to meet the requirement of clinical detection by using a PCR technology so as to improve the specificity and sensitivity of nucleic acid detection.

Disclosure of Invention

In order to solve the problems, the invention provides a primer probe composition, application thereof in PCR reaction and a nucleic acid detection kit comprising the primer probe composition, so that the technical effects of improving the specificity of nucleic acid detection and reducing false positive are realized, and the primer probe composition is particularly suitable for application in detection kits of mutant sequences with high requirements on the specificity of primers.

In a first aspect of the invention, there is provided a primer probe composition for nucleic acid sequence detection, comprising: a first upstream primer F1, a second upstream primer F2, a probe P, a downstream primer R and an oligonucleotide F1C;

wherein, the first upstream primer F1 is composed of the following two parts from 5 'end to 3' end in sequence:

(1) a mismatch area: no complementary pairing with the target nucleic acid sequence; the sequence of the mismatch region includes, from the 5 'end to the 3' end, the same sequence as the second upstream primer F2, and the same sequence as the probe P;

(2) a matching area: complementary pairing with a target nucleic acid sequence;

the end of oligonucleotide F1C was modified so that it could not be extended. In some embodiments, the end of the oligonucleotide F1C can be modified in a manner well known in the art to block the 3' -OH so that it cannot be extended in the PCR amplification reaction. Exemplary modifications include phosphorylation modifications, C3 spacer modifications, and modifications with the BHQ1 group, among others.

Oligonucleotide F1C has a partial complementary match of the nucleic acid sequence to the matching region of the first forward primer F1. The complementary paired sequences may or may not include an amplification site.

In some embodiments, the length of the oligonucleotide F1C is 45% to 90% of the length of the matching region of the first forward primer F1, e.g., can be 45%, 47%, 50%, 60%, 70%, 71%, 75%, 80%, 85%, 86%, 88%, 90%, etc., preferably, the length of the oligonucleotide F1C is 70% to 86% of the length of the matching region of the first forward primer F1.

In some embodiments, only one of the reverse primers in the primer probe composition, i.e., the first and second forward primers share a single reverse primer. Preferably, the complementary region of the downstream primer and the target nucleic acid sequence is located within a region of 1-200bp downstream of the complementary region of the first upstream primer and the target nucleic acid sequence, and may be, for example, 1bp, 20bp, 40bp, 70bp, 90bp, 110bp, 130bp, 150bp, 170bp, 180bp, 190bp, 200bp, etc.

In some embodiments, the Tm value of the first forward primer is the same as the Tm value of the second forward primer. In some embodiments, the Tm value of the first forward primer is different from the Tm value of the second forward primer, i.e., the Tm value of the first forward primer may be higher than the Tm value of the second forward primer, or the Tm value of the first forward primer may be lower than the Tm value of the second forward primer. In some preferred embodiments, the Tm of the first upstream primer differs from the Tm of the second upstream primer by 0 ℃ to 20 ℃, and may be, for example, 0 ℃, 5 ℃, 8 ℃, 10 ℃, 12 ℃, 15 ℃, 18 ℃, 20 ℃, or the like. In some preferred embodiments, the Tm of the first forward primer is higher than the Tm of the second forward primer.

In some embodiments, the 3' end of the first upstream primer has an amplification decision site that is complementary to a variation detection site on the target sequence, and the amplification decision site has a mismatch region consisting of more than one base upstream of the target sequence that is not complementary to the target sequence; in some embodiments, the mismatch region is 1-15 bases in length, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 bases; in some embodiments, the mismatch region is located 2 to 14 bases from the 3' end of the first forward primer.

In some embodiments, the matching region and the non-matching region of the first forward primer are separated by 1 or more bases, and there may be no base separation. The mismatch region of the first forward primer does not pair identically or complementarily with the target nucleic acid sequence or hybridize under high stringency conditions.

In some embodiments, the probe carries a reporter group. In a preferred embodiment, the reporter group is detectable only after the probe is hydrolyzed. In a further embodiment, the probe carries a reporter group and a quencher group. In still further embodiments, the reporter group can be a fluorophore selected from the group consisting of: FAM, HEX, VIC, ROX, Cy5, Cy3, etc.; the quencher group may be selected from the group consisting of: TAMRA, BHQ1, BHQ2, BHQ3, DABCYL, QXL, DDQI, etc. In some embodiments, the hydrolysis probe does not carry any other modifications besides the reporter and quencher, e.g., MGB, LNA, PNA, BNA, SuperBase, etc. In a preferred embodiment, the probe of the invention is a Taqman probe. In a preferred embodiment, the reporter is located at the 5 'end of the probe and the quencher is located at the 3' end of the probe.

In some embodiments, the first upstream primer F1 may be 50-120 bases in length, preferably 50-90 bases in length, preferably 30-70 bases in length. In some embodiments, the Tm of the target sequence binding region of the first forward primer F1 can be 40 ℃ to 90 ℃, preferably 50 ℃ to 80 ℃, with a GC content of 40% to 80%.

In some embodiments, the second upstream primer F2 may be 10-40 bases in length, preferably 13-30 bases in length. In some embodiments, the Tm of the second upstream primer F2 can be 35 ℃ to 85 ℃, preferably 50 ℃ to 80 ℃, and the GC content can be 40% to 80%.

In some embodiments, probe P is 12-30 bases in length. In a further embodiment, probe P has a Tm of 50 ℃ to 80 ℃. In still further embodiments, the GC content of the probe is 40% to 80%.

In some embodiments, the reverse primer R is 15-30 bases in length. In a further embodiment, the reverse primer R has a Tm of 50 ℃ to 80 ℃. In a further embodiment, the reverse primer R has a GC content of 40% to 80%.

In some embodiments, oligonucleotide F1C is 15-30 bases in length. In a further embodiment, the Tm of the oligonucleotide F1C is from 50 ℃ to 80 ℃. In a further embodiment, the GC content of the oligonucleotide F1C is between 40% and 80%.

In a second aspect of the present invention, there is provided an application of the primer probe composition in a PCR reaction. The PCR reaction comprises fluorescent quantitative PCR and digital PCR.

In a third aspect of the present invention, there is provided a nucleic acid detection kit comprising the primer probe composition, wherein the detection kit is particularly suitable for detecting a mutation sequence requiring high primer specificity, for example, a mutation detection kit for exon 20 insertion of EGFR gene comprising the primer probe composition, and a mutation detection kit for T790M EGFR gene comprising the primer probe composition.

In a fourth aspect of the present invention, there is provided a method for using a nucleic acid detection kit comprising the above primer probe composition, comprising:

mixing a sample to be detected with the primer probe composition and the amplification reagent to obtain a sample mixture;

denaturing the double-stranded DNA in the sample mixture into single strands;

randomly distributing the sample mixture into more than 500 reaction units, wherein each reaction unit contains 1 single strand or does not contain a single strand;

carrying out uniform thermal cycle amplification on all reaction units;

detecting the presence of a single strand in each reaction unit;

the primer probe composition comprises a first upstream primer F1, a second upstream primer F2, a probe P, a downstream primer R and an oligonucleotide F1C, and is the primer probe composition provided by the first aspect of the invention.

The "amplification reagent" refers to a reagent for PCR, including but not limited to dNTPs, DNA polymerase, and some reagents for promoting PCR reaction, such as KCl, MgCl₂Tris-HCl, Dithiothreitol (DTT), and the like.

The purpose of the above-mentioned "denaturation" is to break the hydrogen bonds between pairs of complementary bases on double-stranded DNA, thereby allowing the double strand to separate into two single strands. In some embodiments, single strands can be formed by heating a mixture containing double stranded DNA, for example, heating the mixture to 90 ℃, 92 ℃, 95 ℃, or 98 ℃ to dissociate double stranded DNA. Typically, upon double-stranded dissociation, the mixture needs to be maintained at the elevated temperature for at least 10 seconds or more, e.g., 30 seconds, 1 minute, 2 minutes, 5 minutes, or even more, to achieve 90% or more dissociation of the double-stranded DNA. After dissociation is complete, it is cooled to room temperature or below.

In some embodiments, the above "denaturation" can also be accomplished by altering the solution ionic strength (e.g., adding acids, bases, salts, etc.) to break hydrogen bonds between double-stranded DNA, and an enzyme (e.g., helicase) can also be employed to effect dissociation of double-stranded DNA into single-stranded DNA.

In some embodiments, the above-described "thermocycling amplification" includes the following: pre-amplifying the target nucleic acid sequence by taking the first upstream primer (F1) and the downstream primer (R) as primers to obtain a pre-amplification product; and continuing amplification of the pre-amplification product by using the second upstream primer (F2) and the downstream primer (R) as primers. In some embodiments, F1 and R are pre-amplified as primers for 3-10 cycles; more preferably, the number of cycles of pre-amplification is 5-8 cycles. In some embodiments, F2 and R are used as primers for amplification while the probe (P) is hydrolyzed for a number of cycles ranging from 35 to 50, more preferably 40 to 45.

In some embodiments, the pre-amplification is 5 ℃ to 20 ℃ higher, more preferably 10 ℃ to 15 ℃ higher, than the annealing temperature for continued amplification.

The procedures and reaction conditions (e.g., denaturation temperature, time, etc.) used for pre-amplification and for continued amplification are well known in the art. For example, in some exemplary embodiments, the specific reaction conditions for pre-amplification and continued amplification may be: pre-denaturation at 92-96 deg.C for 5-15 min; denaturation at 92-95 deg.C for 10-60 s, annealing at 55-75 deg.C and extension for 30-90 s for 3-10 cycles; denaturation at 92-95 deg.C for 10-60 s, annealing at 45-65 deg.C and extension for 30-90 s for 35-50 cycles; inactivating at 94-98 deg.c for 5-15 min; the reaction is terminated at 4-15 ℃.

The concentration of primers and probes in the amplification reaction system of the present invention can be determined by routine experimentation in the art. In some exemplary embodiments, the concentration of F1 primer in the reaction system is 15nM-150nM, the concentration of F2 primer is 150nM-1500nM, the concentration of probe P is 50nM-800nM, the concentration of R primer is 150nM-1800nM, and the concentration of oligonucleotide F1C is 15nM-500 nM. In some more preferred embodiments, the concentration of F1 primer in the reaction system is 100nM to 200nM, the concentration of F2 primer is 300nM to 600nM, the concentration of probe P is 150nM to 400nM, the concentration of primer R is 300nM to 900nM, and the concentration of oligonucleotide F1C is 50nM to 300 nM.

The "detection of the presence of a single strand in each reaction unit" in the present invention may employ the principle of the TaqMan probe method, i.e., hydrolysis of a probe by the 5 '-3' exonuclease activity of DNA polymerase to generate a fluorescent signal. If the reaction unit contains the target nucleic acid to be detected, the probe is specifically combined with the target nucleic acid, and a fluorescent signal is emitted in the thermal cycle amplification process. In the invention, the principle of a TaqMan probe method can be adopted for counting the reaction units with single strands, and each reaction unit contains 1 single strand or does not contain a single strand, so that the number of the reaction units emitting fluorescent signals represents the number of target nucleic acids in a reaction system, and finally the concentration of the target nucleic acids in a sample to be detected can be obtained through Poisson correction.

In some embodiments, the sample to be tested can be a biological sample, such as a biological fluid, a living tissue, a frozen tissue, a paraffin section, and the like. In some preferred embodiments, the sample is, e.g., peripheral blood, urine, lavage, cerebrospinal fluid, stool, saliva, and the like.

The method for detecting a nucleic acid sequence provided by the present invention may be a method for detecting a certain nucleic acid in a sample to be detected, for example, only a mutant target sequence or only a wild-type target sequence in the sample, in which case, a first upstream primer and a probe may be used in an amplification reaction system. The detection method may also be used for detecting two or more nucleic acids, for example, simultaneously detecting one mutant nucleic acid and wild-type nucleic acid, or simultaneously detecting two or more mutant nucleic acids and wild-type nucleic acids, and the like, in which case, a plurality of (e.g., 2 or more) first upstream primers (e.g., a plurality of first upstream primers in the same reaction unit), and a plurality of (e.g., 2 or more) probes (e.g., a plurality of probes in the same reaction unit) may be used in the same amplification reaction system. For example, the plural first forward primers differ in their matching regions, and the plural probes have mutually different sequences and reporter genes. Optionally, the mismatch regions of the plurality of first forward primers may also be different. In some embodiments where multiple first forward primers and probes are used, different first forward primers may share the same downstream primer.

In some embodiments, the methods of nucleic acid sequence detection provided herein can be used to detect variations in nucleic acid sequences. The nucleic acid sequence variation may be one or more selected from the group consisting of: base substitution mutations, insertion mutations, deletion mutations, and inversion mutations. In some embodiments, the nucleic acid sequence variation is a base substitution mutation, i.e., there is no difference in the number of bases between the two target sequences (e.g., wild type and mutant) to be detected, e.g., where one or more bases are of a different type. In some embodiments, the nucleic acid sequence variation is a point mutation, i.e., only 1 base type is different.

Definition of

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

As used herein, the terms "oligonucleotide", "polynucleotide" are used interchangeably and refer to single-and/or double-stranded polymers of nucleotide monomers, including, but not limited to, 2' -Deoxyribonucleotides (DNA) and Ribonucleotides (RNA) linked by internucleotide phosphodiester linkages or internucleotide analogs. Nucleotide monomers in nucleic acids may be referred to as "nucleotide residues". Nucleic acids can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof, and can include nucleotide analogs. The nucleotide monomeric units may comprise any of the nucleotides described herein, including (but not limited to) nucleotides and/or nucleotide analogs. Nucleic acids typically range in size from a few nucleotide residues to several thousand nucleotide residues. Wherein "oligonucleotide" generally refers to a polymer of nucleotides of relatively short length, e.g., 1-80. Unless otherwise indicated, whenever a nucleic acid sequence is presented, it is understood that nucleotides are in 5 'to 3' order from left to right. Unless otherwise noted, "a" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, "T" represents deoxythymidine, and "U" represents deoxyuridine.

According to the customary terminology in the art, the length of a nucleic acid can be expressed as bases, base pairs (abbreviated "bp"), nucleotides/nucleotide residues (abbreviated "nt"), or kilobases ("kb"). The terms "base", "nucleotide residue" may describe a polynucleotide, either single-stranded or double-stranded, where the context permits. When the term is applied to double-stranded molecules, it is used to refer to the entire length and should be understood as equivalent to the term "base pair".

The term "mismatched base" refers to a base pair in the double helix structure of DNA which is not an arbitrary base, and is always the principle that adenine (A) is paired with thymine (T) and guanine (G) is paired with cytosine (C). If A is paired with C or G, or T is paired with G or C, it is a base mismatch.

The term "primer" denotes an oligonucleotide: which hybridizes to a sequence in a target nucleic acid and is capable of serving as a point of initiation of complementary strand synthesis (under conditions suitable for such synthesis) along the nucleic acid.

The term "probe" as used herein denotes an oligonucleotide that: which hybridizes to a sequence in the target nucleic acid and is typically detectably labeled. The probes may have modifications (such as 3 '-terminal modifications and/or 5' -terminal modifications that allow the probes to be detected or hydrolyzed by nucleic acid polymerases, etc.) that may also include one or more chromophores.

As used herein, the terms "target sequence", "target nucleic acid sequence" or "target" are used interchangeably and refer to the portion of a nucleic acid sequence to be amplified, detected or amplified and detected, which can anneal or hybridize to a probe or primer under hybridization, annealing or amplification conditions. The term "hybridization" refers to a base pairing interaction between two nucleic acids that results in the formation of a duplex. It is not required that 2 nucleic acids have 100% complementarity over their entire length to achieve hybridization.

The terms "matching region", "target sequence binding region", as used herein, are used interchangeably and refer to a region that is complementary paired with a target nucleic acid sequence.

The term "forward primer", also referred to as "forward primer", as used herein, is an oligonucleotide that extends uninterrupted along the negative strand; the term "downstream primer", also called reverse primer, as used herein, is an oligonucleotide that extends uninterrupted along the forward strand. The positive strand, i.e., the sense strand, also called the coding strand, is generally located at the upper end of the double-stranded DNA in the direction from left to right 5 '-3', and the base sequence is substantially identical to the mRNA of the gene; the primer binding to the strand is a reverse primer; the negative strand, i.e., the nonsense strand, is also called the noncoding strand, is complementary to the positive strand, and the primer that binds to this strand is the forward primer. It is understood that when the designations of sense and antisense strands are interchanged, the corresponding forward and reverse primer designations may be interchanged accordingly.

In the context of the present invention, "first forward primer F1", "first forward primer F1", "first forward primer" are used interchangeably; "second forward primer F2" and "second forward primer F2" are used interchangeably; "reverse primer" and "reverse primer R" are used interchangeably; "Probe P" and "probe" are used interchangeably; "F1C" and "oligonucleotide F1C" are used interchangeably.

As used herein, the terms "upstream," "at/upstream of … …," "… …" and the like, in the context of describing nucleic acid sequences, refer to a portion of the same nucleic acid sequence that is closer to the 5' end than a reference region, e.g., can be immediately adjacent to the reference region or can be separated from the reference region by one or more bases. As used herein, the terms "downstream," "at/downstream of … …," "having … …" and the like, in the context of describing nucleic acid sequences, refer to portions of the same nucleic acid sequence that are 3' of the referenced region, e.g., can be immediately adjacent to the referenced region or can be separated from the referenced region by one or more bases. It will be appreciated that, unless otherwise indicated, where the nucleic acid being described is a double-stranded nucleic acid, the designations "upstream" and "downstream" are generally based on the 5 'and 3' ends of the sense strand.

The terms "Taqman probe (TaqMan probe)" and "hydrolysis probe (hydrolysis probe)" are used interchangeably herein. The Taqman probe is a fluorescence detection technology developed on a Real-time PCR technology platform, the 5 'end of the probe contains a fluorescence reporter group, and the 3' end of the probe contains a fluorescence quenching group. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group, and when PCR amplification is carried out, the exonuclease activity from the 5 'end to the 3' end of Taq DNA polymerase enzyme cuts and degrades the probe, so that the reporter group and the quenching group are separated, and the fluorescent signal is emitted, thereby achieving the complete synchronization of the accumulation of the fluorescent signal and the formation of a PCR product. Specifically, the reporter group may be FAM, HEX, VIC, ROX, Cy5, Cy3, or the like, and the quencher group may be TAMRA, BHQ1, BHQ2, BHQ3, DABCYL, QXL, or DDQI, but not limited thereto. In addition, other modification forms are derived from the Taqman probe, for example, the Taqman-MGB probe is a Taqman probe with minor groove binding Molecules (MGBs) at the 3' end, the Tm value of the probe is improved, the length of the probe is shortened, and simultaneous detection of multiple mutation sites is facilitated.

The "stringent conditions" described herein may be any of low stringency conditions, medium stringency conditions and high stringency conditions. "Low stringency conditions" such as 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 50% formamide, 32 ℃; further, "medium stringent conditions" include, for example, conditions of 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 50% formamide, 42 ℃; "high stringency conditions" include, for example, 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 50% formamide, and 50 ℃ conditions. Under these conditions, it is expected that a polynucleotide having a high homology, such as DNA, can be obtained more efficiently at a higher temperature. Although there are various factors that affect the stringency of hybridization, such as temperature, probe concentration, probe length, ionic strength, time, salt concentration, etc., one skilled in the art can obtain similar stringency by appropriately selecting these factors.

The term "mutation abundance" as used herein refers to a relative or absolute quantitative value of the mutant target gene, and is generally defined as the ratio of the number of mutant target gene molecules to the total number of DNA molecules in the assay.

The term "test sample" as used herein means any composition containing nucleic acid or suspected of containing nucleic acid. The sample may be derived from a biological source ("biological sample"), such as tissue (e.g., biopsy sample), extracts or cultures, and biological or physiological fluids, among others. For example, samples may include skin, plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs, and tumors. Also, samples may include samples of in vitro cultures established from cells taken from an individual or immobilized samples, such as formalin-fixed paraffin-embedded tissue (FFPET) and nucleic acid isolates isolated therefrom.

Advantageous effects

Compared with the prior art, the primer probe composition and the method have the advantages that:

(1) less false positive and high specificity: according to the primer probe composition provided by the invention, the oligonucleotide F1C with a specific length is introduced to be complementary with the matching region of the upstream primer F1, so that false positive signals can be well avoided in the PCR reaction process, and for target detection of some mutant types with smaller sequence difference or similar sequences to wild types, the primer probe composition can effectively improve the specificity of the primers, and is particularly suitable for mutation detection by using a digital PCR method.

(2) The target nucleic acid sequence is short: the primer probe composition provided by the invention has only two parts which are actually paired and combined with a target nucleic acid sequence: the 3' target sequence binding region of the first forward primer F1 and the sequence of the downstream primer R. Compared with at least three parts in a TaqMan probe method and an ARMS method which are matched with a target nucleic acid sequence (an upstream primer, a probe and a downstream primer), the primer-probe composition provided by the invention has shorter requirement on the length of a target nucleic acid fragment to be detected. The advantage can be embodied in different detection scenes, for example, in the detection of highly fragmented free DNA, since the fragmentation of DNA is random, shorter detection fragments can detect more DNA targets, and thus the detection sensitivity is greatly improved.

(3) The requirements on the target nucleic acid sequence are low: similar to the above advantages, the primer probe design method provided by the present invention has the following advantages that since the probe P can be coupled with the complementary sequence at the 5' end of the upstream primer F1 after the pre-amplification is completed, the actual portion coupled with the target nucleic acid sequence has only two portions: the 3' end sequence of F1 and the sequence of the downstream primer R. Therefore, when a complex target nucleic acid sequence is detected, the design method of the primer probe can avoid a GC unbalanced area, and is particularly lower than a TaqMan probe method and ARMS in the design difficulty of the probe.

(4) The requirement on the content of target DNA in a sample is low: when analyzing samples with the target DNA content lower than the optimal quantity, the invention can be used for effectively increasing the quantity of target sequences in the samples and improving the detection sensitivity, thereby reducing errors caused by too small quantity of samples. Such samples typically include plasma (cf DNA samples), biopsy punctures, or FFPE samples. This method typically involves melting double-stranded DNA into its constituent strands, such as single-stranded DNA (ssdna), prior to droplet formation, and then separating each strand prior to amplification and counting.

(5) The application range is wide: the primer probe composition and the detection method provided by the invention can detect short-fragment DNA smaller than 200bp, have good tolerance to PCR inhibitors, and can be suitable for nucleic acid detection of various sample types, including formalin-fixed paraffin-embedded tissue (FFPE) samples, fresh tissue samples, peripheral blood samples, urine samples, lavage fluid samples, cerebrospinal fluid samples, artificially cultured cell line samples, artificially synthesized plasmid samples and the like.

(6) The sample consumption is less: the primer probe composition provided by the invention can be used for simultaneously detecting the mutant type and wild type target nucleic acid sequences in one reaction tube, can carry out absolute quantification and mutation abundance statistics on the mutant type and wild type target nucleic acid sequences when being applied to digital PCR, and is particularly suitable for detecting rare samples, such as peripheral blood circulation tumor DNA samples.

(7) The cost is low: the primer probe composition provided by the invention does not need expensive MGB modification or LNA modification, greatly reduces the use cost of the primer probe, has better detection performance and meets the requirement of clinical use; particularly, when the primer probe composition provided by the invention is applied to digital PCR, after a target DNA double strand is uncoiled into a single strand, the unbalanced free DNA is not required to be repaired by using a terminal repair enzyme, so that the detection cost is reduced.

(8) The experimental steps are simple and convenient: when the primer probe composition provided by the invention is applied to digital PCR, after a reaction system containing a template is subjected to a melting step, a terminal repair enzyme does not need to be added into a reaction solution for repair, so that the experimental steps are reduced, and the experimental time is saved.

Drawings

FIG. 1 is a graph showing the results of detection of EGFR 20 exon insertion mutation in example 1;

FIG. 2 is a graph showing the results of detection of EGFR 20 exon insertion mutation using oligonucleotide F1C of different lengths in example 2;

FIG. 3 is a graph showing the results of detection of mutation in EGFR gene T790M in example 3.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

The main experimental equipment and materials are as follows:

device/Material	Manufacturer/model
		2X ddPCR Supermix for Probes	Bio-Rad, item number 1863010
Micro-droplet generating card	Bio-Rad, cat # 1864008
		ddPCR microdroplet generating oil	Bio-Rad, cat # 1863005
Sealing strip	Bio-Rad, cat # 1863009
		Micro-droplet analysis of oil	Bio-Rad, cat # 1863004
96-hole plate with half skirt edge	Eppendorf, cat # 30128575
		Droplet generator	Bio-Rad, cat # 1864002
Film sealing instrument	Bio-Rad, cat # 1814000
		PCR thermal cycler	Bio-Rad, cat # 1851197
Droplet analyzer	Bio-Rad, cat # 1864003
		Colo 205 cell line	Kobaibio, cat number CBP-60026
Primer and method for producing the same	Is biosynthesized from living organisms
		Probe needle	Is biosynthesized from living organisms
Nucleic acid fragmenting enzyme (KAPA Frag Kit)	Roche, cat # 7962495001
		Analysis software	Bio-Rad, QuantaSoft digital PCR analysis software

Example 1

In this example, a common nucleic acid variation was taken as an example to test the primer probe composition and the detection system of the present invention. Specifically, EGFR 20 exon insertion mutation was used as an example to simulate clinical samples to evaluate the performance of the assay system of the present invention.

1. Sample preparation:

a fragmented 293T cell line DNA sample was prepared, quantified by a Qubit fluorometer, and diluted to 10 ng/. mu.L to serve as a negative control sample.

Meanwhile, an artificially synthesized plasmid sample containing an EGFR gene 20 exon insertion mutation (COSM12377) sequence was prepared, and after fragmentation, the plasmid sample was diluted and mixed with a negative sample to obtain a positive sample.

Tirs-EDTA buffer without any DNA was used as a blank sample.

2. Preparation of the reaction System

2.1 primer Probe composition

The primer and the probe of the invention are prepared by synthesis of biological engineering GmbH. Specific sequences of the primer probe compositions are shown in Table 1.

Table 1:

in the above primer probe, the first forward primer F1(SEQ ID NO:1) has a total length of 63bp, 21 bases at the 3 'end of the primer are complementarily paired with a target nucleic acid sequence, and 3 bases at the 3' end are amplification determining points of insertion mutation of EGFR gene 20 exon. The oligonucleotide F1C-15(SEQ ID NO:5) has a full length of 15bp and can complementarily pair with 15bp at the 3' end of the target sequence binding region of the first forward primer, and the binding specificity of F1 and the target sequence can be increased. The second forward primer F2(SEQ ID NO:2) has a full length of 19bp, and 19 bases at the 5' end of the first forward primer F1 have the same base sequence as that of the second forward primer F2(SEQ ID NO: 3). The 22 nd to 40 th base sequences at the 5' end of the first forward primer F1 were identical to the base sequence of probe P (SEQ ID NO: 3). After the first forward primer F1 specifically amplifies the target nucleic acid, the amplification product is generated with the base sequence from the 5' end of the F1 primer and its complementary sequence, then the second forward primer F2 and the probe P can be complementarily paired with the corresponding template, and the probe P is hydrolyzed by the extension of F2 to emit a fluorescent signal. The fluorescent signal of the internal reference gene is generated by amplifying an internal reference upstream primer rF and an internal reference downstream primer rR and hydrolyzing an internal reference probe rP.

2.2 reaction System

The contents of the respective reagent components in the PCR reaction solution are shown in Table 2.

Table 2:

2.3 reaction Unit preparation

Melting the prepared PCR reaction solution into single strands by adopting a high temperature of 95 ℃ for one minute, and then rapidly cooling to keep the single strands in a single-strand state;

adding 20 mu L of the melted PCR reaction solution into a sample hole of the microdroplet generation card, then adding 70 mu L of microdroplet generation oil into an oil hole of the microdroplet generation card, and finally sealing the microdroplet generation card by using a sealing strip;

the prepared droplet generation card is placed into a droplet generator and droplet generation is initiated. After about 2 minutes, the droplet preparation is complete, the card slot is removed, and about 40 μ L of the droplet suspension is carefully transferred from the uppermost row of wells to a 96-well PCR plate.

3. Amplification reading

And (3) sealing the membrane of the 96-well plate, and then placing the 96-well plate in a PCR thermal cycler for PCR amplification. The procedure used for the reaction system was: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 seconds, annealing and extension at 65 ℃ for 60 seconds, and 5 cycles in total; denaturation at 94 ℃ for 30 seconds and annealing and extension at 52 ℃ for 60 seconds for 40 cycles; inactivating at 98 ℃ for 10 minutes; the reaction was terminated at 10 ℃.

After the PCR amplification is finished, the 96-well plate is placed in a microdroplet analyzer to select FAM/HEX channel for signal reading.

4. Analytical statistics

The intensity of the fluorescence signal and the negative and positive determinations were analyzed using QuantaSoft analysis software, and the results are shown in fig. 1.

In fig. 1, no false positive signal was generated in the blank control and the negative control, i.e., the negative match rate was 100%; signals are gathered in the detection of the positive samples, and the threshold division is clear. Therefore, the primer probe composition has high detection accuracy and specificity in nucleic acid detection application.

Example 2

To demonstrate the effect of the present invention, this example uses different lengths of oligonucleotide F1C to evaluate the effect on detection specificity.

1. Sample preparation:

Meanwhile, an artificially synthesized plasmid sample containing an EGFR gene 20 exon insertion mutation and another mutant type sequence (COSM12378) is prepared, and after fragmentation treatment, a negative sample is used for dilution and mixing to serve as a positive sample.

2. Preparation of the reaction System

2.1 primer Probe composition

The specific sequences of the primer probe compositions are shown in Table 3 below.

Table 3:

in the above primer probe, the oligonucleotide F1C-2-12 has a length of 12bp and can complementarily pair with 12bp at the 3' end of the target sequence binding region of the first forward primer (F1-2); the oligonucleotide F1C-2-15 has a length of 15bp and can complementarily pair with 15bp at the 3' end of the target sequence binding region of the first forward primer (F1-2); the oligonucleotide F1C-2-18 has a length of 18bp and is capable of complementary pairing with 18bp at the 3' -end of the target sequence binding region of the first forward primer (F1-2); the oligonucleotide F1C-2-21 has a length of 21bp and is capable of complementary pairing with the full length of the target sequence binding region of the first forward primer (F1-2).

2.2 reaction System

The contents of the respective reagent components in the PCR reaction solution are shown in Table 4.

Table 4:

in addition, a control group was also set: the PCR reaction solution contained no oligonucleotide F1C.

2.3 reaction Unit preparation

3. Amplification reading

4. Analytical statistics

The intensity of the fluorescence signal and the negative and positive determinations were analyzed using QuantaSoft analysis software, and the results are shown in fig. 2.

In FIG. 2, there was a large number of false positive signals in the negative control (E07) in the reaction system without the addition of oligonucleotide F1C; in the reaction system with the addition of 12bp oligonucleotide F1C-2-12 and 15bp oligonucleotide F1C-2-15, the false positive signals in the negative control (D02 and D06) are obviously reduced, while in the reaction system with the addition of 18bp oligonucleotide F1C-2-18, the false positive signals in the negative control (E01) are basically disappeared, the signals of the positive sample (E02) are still aggregated, the threshold division is clear, and the negative and positive judgment can be better assisted. However, in the reaction system in which the 21bp oligonucleotide F1C-2-21 was added, although there was no false positive signal in the negative control (E04), the signal in the positive reaction (E05) was also suppressed, so that the threshold division was unclear and it was difficult to determine whether the reaction was negative or positive. Therefore, the oligonucleotide F1C can effectively inhibit the generation of false positive signals, improve the specificity of detection, and meanwhile, the oligonucleotide F1C with a specific length can also obviously improve the accuracy of detection.

Example 3

To further verify the effectiveness of the primers and methods of the present invention, another genetic variation (T790M mutation in EGFR gene) was examined.

1. Sample preparation:

Meanwhile, artificially synthesized plasmid samples containing EGFR gene 20 exon T790M mutation (COSM6240) sequences are prepared, and after fragmentation treatment, negative samples are diluted and mixed to serve as positive samples.

Tirs-EDTA buffer without any DNA was used as a blank sample.

2. Preparation of the reaction System

2.1 primer Probe composition

The specific sequences of the primer probe compositions are shown in Table 5 below.

Table 5:

the primers and the probes are synthesized and prepared by the biological engineering corporation.

In the primer probe, the length of the target sequence binding region of the first forward primer F1 is 19bp, the length of the oligonucleotide F1C is 17bp, and the 5 'end of the oligonucleotide F1C can be complementarily paired with the sequence of 9bp at the 5' end of the target sequence binding region of the first forward primer F1.

2.2 reaction System

The contents of the respective reagent components in the PCR reaction solution are shown in Table 6.

Table 6:

2.3 reaction Unit preparation

Same as in example 1.

3. Amplification reading

And (3) sealing the membrane of the 96-well plate, and then placing the 96-well plate in a PCR thermal cycler for PCR amplification. The procedure used was: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 seconds and annealing and extension at 56 ℃ for 60 seconds for 48 cycles; heat treatment at 98 deg.c for 10 min; the reaction was terminated at 10 ℃.

4. Analytical statistics

The intensity of the fluorescence signal and the negative and positive determinations were analyzed using QuantaSoft analysis software. The results are shown in FIG. 3.

In FIG. 3, there was a small false positive signal in the negative control in the reaction system without the addition of oligonucleotide F1C; in a reaction system added with an oligonucleotide F1C with the length of 17bp (wherein 9bp can be complementary with a target sequence binding region), false positive signals in a negative control are obviously reduced and basically disappear, signals of a positive sample still gather, the threshold division is clear, and the negative and positive judgment can be better carried out.

In summary, it can be seen from the above examples that the presence of oligonucleotide F1C significantly reduces the generation of false positive signals, probably due to: oligonucleotide F1C competitively inhibited the complementary pairing of the first upstream primer F1 with the target nucleic acid sequence. Further, when the length of the oligonucleotide F1C is 45% -90% of the length of the target sequence binding region of the first upstream primer, the oligonucleotide F1C can not only effectively reduce the false positive signal in the negative control, but also has low or no influence on the target nucleic acid positive signal in the positive reaction, so that the threshold division is clear, and more effective help in negative-positive determination is achieved.

Furthermore, from the disclosure of the present specification, particularly the detailed description, those skilled in the art can reasonably expect: the primer probe composition is not only suitable for detecting EGFR 20 exon insertion mutation or EGFR gene T790M mutation, but also can improve the detection specificity in the aspect of other mutation detection of EGFR or mutation detection of other genes. Furthermore, although the embodiments of the present application employ digital PCR detection methods, it is reasonably expected by those skilled in the art that the primer probe compositions of the present invention are also suitable for other PCR detection, such as fluorescent quantitative PCR.

Sequence listing

<110> Mike Bio-Ltd

<120> composition for nucleic acid detection

<130> 2020

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

actgctctaa cctccttcca tctcctcacg ctcgctctcc tcagcgtgga caacccccac 60

cac 63

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

actgctctaa cctccttcc 19

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctcctcacgc tcgctctcc 19

<210> 4

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ccagcaggcg gcacac 16

<210> 5

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtggtggggg ttgtc 15

<210> 6

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cctccagagg atgttcaa 18

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcacataggt aatttccaaa 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcccaaggac cacctcacag 20

<210> 9

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

actgctctaa cctccttcca tctcctcacg ctcgctctcc tcgatggcca gcgtggacgg 60

taa 63

<210> 10

<211> 12

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ttaccgtcca cg 12

<210> 11

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ttaccgtcca cgctg 15

<210> 12

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ttaccgtcca cgctggcc 18

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ttaccgtcca cgctggccat c 21

<210> 14

<211> 61

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ccgtctcgca ttaacatatt atctcctcac gctcggactc gaccaccgtg cagctcagca 60

t 61

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ccgtctcgca ttaacatatt 20

<210> 16

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ctcctcacgc tcggactcg 19

<210> 17

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ttcccggaca tagtccag 18

<210> 18

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gcacggtgga ggtgagg 17

Claims

1. A primer probe composition for nucleic acid detection comprising: a first upstream primer F1, a second upstream primer F2, a probe P, a downstream primer R and an oligonucleotide F1C;

the first upstream primer F1 is composed of the following two parts from 5 'end to 3' end in sequence:

(2) a matching area: complementary pairing with a target nucleic acid sequence;

the end of the oligonucleotide F1C is modified so as not to be extendable; the oligonucleotide F1C has a partial complementary pair of nucleic acid sequences with the matching region of the first forward primer F1.

2. The primer probe composition of claim 1, wherein the oligonucleotide F1C has a length that is 45% -90%, preferably 70% -86% of the length of the matching region of the first upstream primer F1.

3. The primer probe composition of claim 1, wherein the region of complementarity of the reverse primer R to the target nucleic acid sequence is located within a region 1-200bp downstream of the region of complementarity of the first forward primer F1 to the target nucleic acid sequence.

4. The primer probe composition of claim 1, wherein the Tm of the first forward primer F1 is 0-20 ℃ different from the Tm of the second forward primer F2.

5. The primer probe composition of claim 1, wherein the 3' end of the first upstream primer F1 has an amplification decision site that is complementary to a mutation detection site on the target sequence, and a mismatch region consisting of one or more bases that is not complementary to the target sequence is located upstream of the amplification decision site; preferably, the 3' end of the first upstream primer contains a mismatch region consisting of 1 to 15 bases and not complementary to the target sequence at 2 to 14 bases.

6. The primer-probe composition of claim 1, wherein the probe P is modified at the 5 'end and the 3' end with a reporter group and a quencher group, respectively.

7. Use of the primer probe composition of any one of claims 1 to 7 in a PCR reaction.

8. The use according to claim 8, wherein the PCR reaction is a fluorescent quantitative PCR or a digital PCR.

9. A nucleic acid detection kit comprising the primer probe composition of any one of claims 1-7.

10. A method of using the nucleic acid detection kit of claim 9, comprising:

mixing a sample to be tested with the primer probe composition and the amplification reagent of any one of claims 1-7 to obtain a sample mixture;

denaturing the double-stranded DNA in the sample mixture into single strands;

carrying out uniform thermal cycle amplification on all reaction units;

detecting the presence of single strands in each reaction unit.