Primer probe combination, reagent and kit for PCR (Polymerase Chain Reaction) detection of nucleic acid

Abstract

The invention provides a primer probe combination for PCR detection of nucleic acid, the primer probe combination comprises a first oligonucleotide, a second oligonucleotide and a detection probe, and the first oligonucleotide and the second oligonucleotide are a forward primer and a reverse primer respectively; the first oligonucleotide comprises an anchoring region which is not complementary to or identical to the target sequence, and a binding region which is complementary to or identical to the target sequence; the detection probe comprises an anchoring binding region which is complementary or the same as the first oligonucleotide anchoring region, and a detection region which is complementary with the target sequence; and the detection probe is labeled with a detection group. The primer probe is simple in design and low in cost. The probe based on the scheme can be designed to be short enough to perform hybridization of shorter sequences, which is helpful for amplification of conserved regions of microorganisms, viruses and the like; meanwhile, the shorter sequence means that the higher resolution capability on the change of the basic group in the sequence can be achieved, and the primer can be used for single-digit basic group difference target nucleic acid in SNP and tumors, especially detection of single basic group mutation.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and particularly to the fields of nucleic acid amplification and detection. Background Art

[0002] Currently, in qPCR and digital PCR, TaqMan probes are mainly used for the detection of target sequences; TaqMan probes are essentially FRET oligonucleotide probes, with a fluorescent reporter group labeled at the 5' end of the probe and a fluorescent quenching group labeled at the 3' end. During the PCR process, by utilizing the 5'-3' exonuclease activity of Taq enzyme, the TaqMan probe bound to the target sequence is hydrolyzed, enabling the fluorescent group to be released and fluorescence signal to be detected, thereby detecting the target sequence; therefore, TaqMan probes are widely used in gene quantitative detection, genotyping, tumor-related gene expression detection, etc.

[0003] The length of TaqMan probes is generally between 25 and 32 bp because the annealing temperature during the PCR process is generally around 60°C; if the probe is too short, the probe Tm will be too low to hybridize to the target sequence, and if it is too long, the probe specificity will decrease. During the detection of certain microbial nucleic acids, due to the short conserved region, shorter probes need to be designed, or degenerate bases need to be introduced; in the detection of gene SNPs or tumor mutations, shorter probes also need to be designed to improve specificity; by modifying some bases with locked nucleic acids, etc., the Tm value can be increased, thereby reducing the probe length and improving specificity; however, the modification will increase the synthesis cost of the probe, and in addition, excessive modification may lead to difficult hydrolysis of the probe and thus a decrease in the generation of fluorescence signals. Summary of the Invention

[0004] Based on this, one of the objectives of the present invention is to provide a primer-probe combination for PCR detection of nucleic acids, and the primer-probe combination includes a first oligonucleotide, a second oligonucleotide, and a detection probe.

[0005] The first oligonucleotide and the second oligonucleotide are a forward primer and a reverse primer to each other.

[0006] The first oligonucleotide includes an anchoring region that is not complementary or identical to the target sequence, and a binding region that is complementary to the target sequence.

[0007] The detection probe includes an anchoring binding region that is complementary or identical to the anchoring region of the first oligonucleotide, and a detection region that is complementary to the target sequence; the detection probe is labeled with a detection group.

[0008] In one embodiment, the anchoring region of the first oligonucleotide is upstream of the binding region.

[0009] In another embodiment, the anchoring binding region of the detection probe has a length of 9 to 16 bases, and the detection region of the detection probe has a length of 9 to 16 bases;

[0010] In another embodiment, the 3'-end of the detection probe is blocked.

[0011] In another embodiment, the 3'-end blocking is selected from one of amino group blocking modification, C3-Spacer modification, thiol modification, dideoxy modification, phosphate group modification, and detection group modification.

[0012] In another embodiment, the detection group includes a fluorescent group and a quenching group. Another object of the present invention is to provide a reagent for PCR nucleic acid detection, and the reagent includes the primer-probe combination described in any one of the above;

[0013] In one embodiment, the reagent further includes dNTP, buffer, salt ions, and polymerase.

[0014] Another object of the present invention is to provide a kit for PCR nucleic acid detection, and the kit includes the primer-probe combination described in any one of the above, or the reagent described above.

[0015] Another object of the present invention is to provide a method for specifically detecting a target nucleic acid with single-digit base differences, including the following steps:

[0016] a) Contacting a sample containing the target nucleic acid with a first oligonucleotide, a second oligonucleotide, and a detection probe;

[0017] The first oligonucleotide and the second oligonucleotide are a forward primer and a reverse primer to each other;

[0018] The first oligonucleotide includes an anchoring region that is not complementary or identical to the target sequence, and a binding region that is complementary to the target sequence;

[0019] The detection probe includes an anchoring binding region that is complementary or identical to the anchoring region of the first oligonucleotide, and a detection region that is complementary to the target sequence; the detection region is labeled with a detection group;

[0020] b) Performing a PCR reaction on the mixture in step a) to form an amplification product;

[0021] c) Specifically detecting the target nucleic acid with single-digit base differences based on the melting analysis or hybridization analysis of the amplification product.

[0022] In one embodiment, it further includes a step of performing a reverse transcription reaction using reverse transcriptase to form a reverse transcription product.

[0023] In another embodiment, when the anchoring binding region of the detection probe is complementary to the first oligonucleotide anchoring region, the concentration of the first oligonucleotide is higher than that of the second oligonucleotide; the concentration ratio of the first oligonucleotide to the second oligonucleotide is 6:6 to 6:1, preferably the concentration ratio is 6:2 to 6:1;

[0024] In another embodiment, when the anchoring binding region of the detection probe is the same as the first oligonucleotide anchoring region, the concentration of the first oligonucleotide is lower than that of the second oligonucleotide; the concentration ratio of the first oligonucleotide to the second oligonucleotide is 1:6 to 6:6; preferably the concentration ratio is 1:6 to 2:6.

[0025] The primer probe of the present invention is simple in design and low in cost. The probe based on the scheme can be designed short enough to hybridize with shorter sequences, which is very helpful for the amplification of conserved regions of microorganisms and viruses, etc.; at the same time, the shorter sequence means that it has a stronger ability to distinguish base changes, and can be used for the detection of target nucleic acids with single-digit base differences in SNPs and tumors, especially the detection of single-base mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figures 1 - 2 Shows the digital PCR detection result of enterovirus in Example 2 of the present invention and the melting curve fitting peak of qPCR;

[0027] Figure 3 Shows the influence of different upstream and downstream primer concentration ratios on detection in the embodiments of the present invention;

[0028] Figure 4 Shows the influence of the complementary length between the anchoring binding region of the detection probe and the primer anchoring region on detection in the embodiments of the present invention;

[0029] Figures 5 - 6 Shows the influence of the length of the detection region of the detection probe on detection in the embodiments of the present invention;

[0030] Figure 7 Shows the influence of the relationship between the anchoring binding region of the detection probe and the primer anchoring region on detection in the embodiments of the present invention;

[0031] Figure 8 Shows the influence of the position where the detection region of the detection probe is set on detection in the embodiments of the present invention;

[0032] Figure 9 Shows the digital PCR detection result of the BRAF gene in Example 9 of the present invention. DETAILED EMBODIMENTS

[0033] Definition

[0034] Unless otherwise defined, 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. The following definitions supplement those in the art and are related to this application, but do not extrapolate to any related or unrelated situations, such as any commonly used patents or applications. Although any methods and materials similar or equivalent to those described herein can be used in testing the invention in practice, the materials and methods described herein are preferred. Thus, the terms used herein are intended to describe only particular embodiments and are not intended to limit the invention.

[0035] In this document, the terms "one or more" and "at least one" are used interchangeably herein.

[0036] Nucleic acids include DNA and RNA and DNA-RNA chimeras can be double-stranded or single-stranded. DNA can be genomic, cDNA, methylated DNA or synthetic DNA. RNA can be mRNA, miRNA, tRNA, rRNA, hnRNA, methylated RNA, and the like. The term "nucleic acid" includes any physical string of monomeric units which can correspond to a string of nucleotides, including polymers of nucleotides (e.g., typical DNA or RNA polymers), peptide nucleic acids (PNAs), modified oligonucleotides (e.g., oligonucleotides containing bases atypical for biological RNA or DNA in solution, e.g., 2'-O-methylated oligonucleotides), and the like. Nucleic acids can be, for example, single-stranded or double-stranded.

[0037] The four traditional nucleobases are A, T / U, C, and G, where T is present in DNA and U is present in RNA. Nucleotides found at the target are typically natural nucleotides (deoxyribonucleotides or ribonucleotides). This is also the case for the nucleotides that form primers.

[0038] The terms "target sequence", "target nucleic acid", "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 to a primer under annealing or amplification conditions.

[0039] The terms "nucleic acid", "polynucleotide" and "oligonucleotide" refer to polymers of nucleotides (e.g., ribonucleotides or deoxyribonucleotides), and include naturally occurring (e.g., adenosine, guanidine, cytosine, uracil, and thymidine) and non-naturally occurring (human co-modified) nucleic acids. The term is not limited by the length of the polymer (e.g., the number of monomers). Nucleic acids can be single-stranded or double-stranded and typically contain 5'-3' phosphodiester bonds, although in some cases, nucleotide analogs may have other bonds. Monomers are typically referred to as nucleotides.

[0040] The term "forward primer", also known as upstream primer, is an oligonucleotide that extends continuously along the negative strand; the term "reverse primer" used in the present invention, also known as downstream primer, is an oligonucleotide that extends continuously along the positive strand. Among them, the positive strand, i.e., the sense strand, the coding strand, is generally located at the upper end of double-stranded DNA, with the direction from left to right being 5'-3', and its base sequence is basically the same as that of the mRNA of this gene; the primer binding to this strand is the reverse primer; the negative strand, i.e., the antisense strand, also known as the non-coding strand, is complementary to the positive strand, and the primer binding to this strand is the forward primer. It should be understood that when the designation of the sense strand and the antisense strand is interchanged, the corresponding naming of the forward and reverse primers can also be interchanged accordingly.

[0041] The term "probe" refers to a labeled oligonucleotide used to detect the presence of a target.

[0042] The terms "complementary" and "base complementarity" refer to the phenomenon in which corresponding relationships such as A with T, A with U, and G with C are connected to each other by hydrogen bonds. Correspondingly, "mismatched bases" refer to all other pairing situations except those specified in "base complementary pairing", such as A with C, A with G, T with G, or T with C mismatches, etc. The term "identical" means that two nucleic acid sequences have the same sequence.

[0043] The so-called blocking, that is, the part used to prevent the extension of the nucleic acid strand by DNA polymerase, thereby preventing strand extension in, for example, the PCR process. The blocking can be modified with Spacer C3, Spacer C8 (Octanediol), Spacer 9 / TEG, Spacer C12, Spacer 18 / HEG, THF (tetrahydrofuran) / dSapcer (abasic site) to block the corresponding sites and thus prevent the extension reaction. The blocking can also be a polymerase enzyme-blocking group, a group with the function of blocking the further extension of the polymer. The blocking group can be any chemical group that can be connected to a nucleotide, which will allow the 5' end of the modified nucleotide to be connected to the 3' end of another nucleotide in the DNA strand, but will not allow the nucleotide to be connected to the 3' hydroxyl group of the modified nucleotide. Appropriately, the absence of the OH group at the 3' position will prevent further extension through polymerase activity.

[0044] The term "sample" refers to a composition that may contain one or more target nucleic acids, including patient samples, plant or animal materials, waste materials, materials for forensic analysis, environmental samples, circulating tumor cells (CTCs), cell-free DNA, liquid biopsy samples, and so on. Samples include any tissue, cell, or extract derived from a living or dead organism that may contain target nucleic acids. For example, biopsy tissue samples including peripheral blood, bone marrow, plasma, serum, lymph nodes, respiratory system tissues or exudates, gastrointestinal tissues, urine, feces, sperm, or other body fluids. Specific target samples are tissue samples (including body fluids) from a human or animal having or suspected of having a disease or disorder (especially infected by a virus). Other target samples include industrial samples, such as samples for water testing, food testing, pollution control, and so on. The components of a sample may include target nucleic acids, non-target nucleic acids, and other materials (e.g., salts, acids, bases, detergents, proteins, carbohydrates, lipids, and other organic or inorganic materials). Samples may or may not undergo a process of purifying the target nucleic acid before amplification. Further processing may involve treatment with a detergent or denaturing agent to release nucleic acids from cells or viruses, removing or inactivating non-nucleic acid components, and concentrating the nucleic acids.

[0045] The term "kit" refers to any manufactured article (e.g., a package or container) that includes at least one reagent (such as a nucleic acid probe or a probe combination, etc.) that is used as described herein for specifically amplifying, capturing, labeling / converting, or detecting RNA or DNA.

[0046] The term "amplification product" refers to the product of an amplification reaction. The amplification product includes the primers used to initiate polynucleotide synthesis in each round. An "amplicon" is a sequence targeted for amplification, and this term can also be used to refer to the amplification product. The 5' and 3' boundaries of an amplicon are defined by the forward primer and the reverse primer. "Reverse transcription product", "RT product", and similar terms refer to cDNA molecules produced by extending an RT primer on an RNA template by a polymerase having reverse transcriptase activity.

[0047] The term "comprising", when preceding a recitation of steps or elements, means the addition of further steps or elements, which are optional and not excluded.

[0048] Embodiment

[0049] In one aspect, the present invention provides a primer-probe combination which, like traditional primer-probe combinations for PCR detection, contains a set of primers (1 forward primer and 1 reverse primer), and 1 detection probe for detecting gene modification.

[0050] In a specific embodiment, the forward primer comprises an anchoring region that is not complementary or identical to the target, and a binding region that is complementary to the target sequence; the detection probe comprises an anchoring binding region that is complementary to the anchoring region of the forward primer, and a detection region that is complementary to the target sequence.

[0051] The anchoring region of the forward primer is upstream of the binding region.

[0052] Furthermore, the anchoring region of the forward primer is complementary to the anchoring binding region of the detection probe by 9 to 16 bases, for example, it can be 9, 10, 11, 12, 13, 14, 15 or 16 bases complementary. The detection region of the detection probe is complementary to the extended strand of the forward primer.

[0053] Furthermore, the detection gene comprises a fluorophore and a quencher.

[0054] There are no particular limitations in the present invention, and any common fluorophores and quenchers in the field of diagnostic reagents can be used. Exemplary fluorophores can be selected from various fluorescent markers, such as one or more of ALEX-350, FAM, VIC, TET, CALFluorGold540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CALFluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705; the quencher can be selected from various quenchers, such as one or more of DABCYL, BHQs (such as BHQ-1 or BHQ-2), ECLIPSE, and / or TAMRA.

[0055] Furthermore, the anchoring binding region of the detection probe is upstream of the detection region. Preferably, the fluorophore or the quencher is modified at the 3'-end of the detection region of the detection probe; more preferably, the fluorophore and the quencher are respectively modified at the 3'-end and 5'-end of the detection region of the detection probe.

[0056] Furthermore, the anchoring binding region of the detection probe is downstream of the detection region. Preferably, the 3'-end of the detection probe is blocked and modified. The present invention has no particular limitation, and any common blocking modification in the field of diagnostic reagents can be used. Exemplary blocking modifications can be that the 3'-OH is modified with 3'-Spacer C3, 3'-Phosphat, 3'-ddC, 3'-InvertedEnd, etc., so that the 3'-OH is blocked, thereby preventing its extension reaction. It can also be a polymerase enzyme-blocking group, a group having the function of blocking the further extension of the polymer. The blocking group can be any chemical group capable of being linked to a nucleotide, which will allow the 5'-end of the modified nucleotide to be linked to the 3'-end of another nucleotide in the DNA strand but will not allow a nucleotide to be linked to the 3'-hydroxyl group of the modified nucleotide. Appropriately, the absence of the OH group at the 3'-position will prevent further extension through polymerase activity. In some embodiments, the blocking group is selected from acetyl group, CH3, glycyl group, leucyl group, and alanyl group. In other embodiments, the blocking group can be in the form of a di- or tri-peptide. More preferably, the 3'-end blocking is selected from one of amino blocking modification, C3-Spacer modification, thiol modification, dideoxy modification, phosphate group modification, and detection group modification.

[0057] In another specific embodiment, the forward primer comprises an anchoring region that is not complementary or identical to the target, and a binding region that is complementary to the target sequence; the detection probe comprises an anchoring binding region that is identical to the anchoring region of the forward primer, and a detection region that is complementary to the target sequence.

[0058] The anchoring region of the forward primer is upstream of the binding region.

[0059] Furthermore, the anchoring region of the forward primer and the anchoring binding region of the detection probe have 9 to 16 bases in common, for example, they can have 9, 10, 11, 12, 13, 14, 15, or 16 bases in common. The detection region of the detection probe is complementary to the extended strand of the reverse primer.

[0060] Furthermore, the detection gene comprises a fluorescent group and a quenching group.

[0061] The present invention has no particular limitation, and any common fluorescent group and quenching group in the field of diagnostic reagents can be used. Exemplary fluorescent groups can be selected from various fluorescent markers, such as one or more of ALEX-350, FAM, VIC, TET, CALFluorGold540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CALFluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705; the quenching group can be selected from various quenchers, such as one or more of DABCYL, BHQs (such as BHQ-1 or BHQ-2), ECLIPSE, and / or TAMRA.

[0062] Furthermore, the anchoring binding region of the detection probe is upstream of the detection region. Preferably, the fluorescent group or the quenching group is modified at the 3'-end of the detection region of the detection probe; more preferably, the fluorescent group and the quenching group are respectively modified at the 3'-end and 5'-end of the detection region of the detection probe.

[0063] Furthermore, the anchoring binding region of the detection probe is downstream of the detection region. Preferably, the 3'-end of the detection probe is blocked and modified. The present invention has no particular limitation, and any common blocking modification in the field of diagnostic reagents can be used. Exemplary blocking modifications can be that the 3'OH is modified with 3'-Spacer C3, 3'-Phosphat, 3'-ddC, 3'-InvertedEnd, etc., so that the 3'OH is blocked, thereby preventing its extension reaction. It can also be a polymerase enzyme-blocking group, a group with the function of blocking the further extension of the polymer. The blocking group can be any chemical group that can be linked to a nucleotide, which will allow the 5'-end of the modified nucleotide to be linked to the 3'-end of another nucleotide in the DNA strand but will not allow the nucleotide to be linked to the 3'-hydroxyl group of the modified nucleotide. Appropriately, the absence of the OH group at the 3'-position will prevent further extension through polymerase activity. In some embodiments, the blocking group is selected from acetyl group, CH3, glycyl group, leucyl group, and alanyl group. In other embodiments, the blocking group can be in the form of a di- or tri-peptide. More preferably, the 3'-end blocking is selected from one of amino blocking modification, C3-Spacer modification, thiol modification, dideoxy modification, phosphate group modification, and detection group modification. Further preferably, the 5'-end of the forward primer is modified with multiple As to prevent the 3'-extension of the complementary sequence of the forward primer.

[0064] On the other hand, the present invention provides a reagent for PCR detection of nucleic acid, comprising the primer-probe combination described above;

[0065] Further, the reagent further comprises dNTP, buffer, salt ions and polymerase.

[0066] Deoxynucleoside triphosphates, i.e., dNTPs, such as dATP, dCTP, dGTP, dTTP, dITP, dUTP, α-thio-dNTP, biotin-dUTP, fluorescein-dUTP, digoxigenin-dUTP, 7-deaza-dGTP can be used. dNTPs are well known in the art and are commercially available.

[0067] Buffers and salts for use in the present invention provide suitable stabilizing pH and ionic conditions for nucleic acid synthesis, such as reverse transcriptase and DNA polymerase activities. A variety of buffer and salt solutions and modified buffers useful in the present invention are known in the art, including reagents not specifically disclosed herein. Preferred buffers include, but are not limited to, TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES, TAPS, PIPES, CAPS. In a preferred embodiment, the provided composition comprises TRIS. In some embodiments, the composition for use in the methods of the present invention comprises a buffer comprising TRIS having a pH of from about 8 to about 9, and in certain preferred embodiments the TRIS is TRIS-HCl (pH 8.0 - 9.0). Preferred salt solutions include, but are not limited to, ammonium sulfate, magnesium chloride, potassium acetate, potassium sulfate, potassium chloride, ammonium chloride, ammonium acetate, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride solution, sodium acetate, lithium chloride, lithium acetate. In a preferred embodiment, the provided composition comprises ammonium sulfate and magnesium chloride. In certain preferred embodiments, the provided composition comprises TRIS-HCl, ammonium sulfate and magnesium chloride.

[0068] The present invention is not particularly limited, and common polymerases in the field of diagnostic reagents can be used. Exemplary polymerases can be polymerases derived from thermophilic bacteria that are well-known in the art. In specific examples, DNA polymerases derived from Thermus aquaticus (U.S. Patents 4,889,818 and 5,079,352) (trade name Taq polymerase), DNA polymerases derived from Thermus thermophilus (WO 91 / 09950) (rTth DNA polymerase), DNA polymerases derived from Pyrococcus furiosus (WO 92 / 9689) (Pfu DNA polymerase, manufactured by Stratagenes), DNA polymerases derived from Thermococcus litoralis (EP-A455430 (trademark Vent): manufactured by New England Biolabs), etc. are commercially available. Among them, the thermostable polymerase derived from Thermus aquaticus is preferred.

[0069] On the other hand, the present invention provides a kit for PCR detection of nucleic acids, comprising the above primer-probe combination or the above reagents.

[0070] On the other hand, the present invention also provides a method for specifically detecting a target nucleic acid with a single-digit base difference, the method comprising the following steps:

[0071] a) contacting a sample containing the target nucleic acid with a forward primer, a reverse primer, and a detection probe;

[0072] The forward primer comprises an anchoring region that is not complementary or identical to the target sequence, and a binding region that is complementary or identical to the target sequence;

[0073] The detection probe comprises an anchoring binding region that is complementary or identical to the anchoring region of the forward primer, and a detection region that is complementary to the target sequence; the detection region is labeled with a detection group;

[0074] b) performing a PCR reaction on the mixture of step a) to form an amplification product;

[0075] c) specifically detecting the target nucleic acid with a single-digit base difference based on the melting analysis or hybridization analysis of the amplification product.

[0076] The single-digit base difference refers to a difference of 1, 2, 3, 4, 5, 6, 7, 8, or 9 bases.

[0077] In a specific embodiment of the present invention, the detection method further comprises a step of performing a reverse transcription reaction using a reverse transcriptase to form a reverse transcription product.

[0078] As used herein, the term "reverse transcriptase" refers to a polypeptide having reverse transcriptase activity. Enzymes having reverse transcriptase activity can be, for example, retroviral reverse transcriptases such as M-MLV reverse transcriptase, Rous sarcoma virus (RSV) reverse transcriptase, human immunodeficiency virus (HIV) reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase, Rous associated virus (RAV) reverse transcriptase, avian leukosis virus (MAV) reverse transcriptase, avian sarcoma-leukosis virus (ASLV) reverse transcriptase, and lentiviral reverse transcriptase, or corresponding mutants, variants, or derivatives thereof having reverse transcriptase activity. As used herein, "mutants, variants, or derivatives" refers to all transformations that can exist or can be generated of a chemical species that still retain the defined chemical activity of that chemical species.

[0079] Further, when the anchoring binding region of the detection probe is complementary to the anchoring region of the forward primer, the concentration of the forward primer is higher than that of the reverse primer. In a preferred embodiment, the concentration ratio of the forward primer to the reverse primer is 6:6 to 6:1, such as 6:6, 6:5, 6:4, 6:3, 6:2, 6:1; preferably the concentration ratio is 6:2 to 6:1.

[0080] In another specific embodiment, when the anchoring binding region of the detection probe is the same as the anchoring region of the forward primer, the concentration of the forward primer is lower than that of the reverse primer. In a preferred embodiment, the concentration ratio of the forward primer to the reverse primer is 1:6 to 6:6, such as 1:6, 2:6, 3:6, 4:6, 5:6, 6:6; preferably the concentration ratio is 1:6 to 2:6.

[0081] In the present invention, the forward primer and the reverse primer can be interchanged.

[0082] Compared with the prior art, the technical solution of the present invention has the following advantages: First, the probe can be designed short enough for hybridization of shorter sequences, which is very helpful for amplification of conserved regions of microorganisms and viruses, etc.; at the same time, the shorter sequence means stronger resolution ability for base changes therein, and can be used for SNP and single-base mutation detection in tumors; second, by changing the photographing temperature, the hybridization of the probe with the target sequence can be very precisely controlled to achieve the purpose of allowing only certain sequences to hybridize; then, since the probe mainly emits fluorescence by hybridizing with the target sequence and does not need to consider the enzymatic cleavage effect of Taq enzyme, the design of the probe is simpler.

[0083] The following are the preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that for those skilled in the art, based on the inventive concept of this invention, several modifications and improvements made all fall within the protection scope of the present invention. Reagents without indicating the manufacturer can all be obtained as conventional products through commercial purchase.

[0084] In the present invention, the plasmids and primer-probe sets used are all synthesized and provided by General Biology (Anhui) Co., Ltd. qPCR detection is carried out using the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd.; digital PCR detection is carried out using the D600 digital PCR instrument of Mike Bio.

[0085] Enterovirus (EV), also known as enteric virus, is a positive-strand single-stranded RNA virus mainly parasitizing in the intestine. Enterovirus 71 (EVA71) that has been prevalent in Asia and North America in recent years and can cause hand, foot, and mouth disease is one of them. Enterovirus is a type of virus containing many types. Its 5-UTR region is the conserved region of the virus, and probes for the conserved region can be set in this region. However, the length of the conserved region is generally short, and there are also sporadic mutation sites distributed in it; in the design of the enterovirus universal probe, the probe spans 15 bases of the conserved region, and among these 15 bases, there are often 1 to 3 base differences. The following Table 1 shows the PCR reaction system for detecting EVA71, and the total volume of the entire reaction system is 25 μL.

[0086] Table 1

[0087] Reagent components Concentration 2×PCR reaction buffer 1× Reverse transcriptase 3U DNA polymerase 3U Probe 250 nM Forward primer 500 nM Reverse primer 120 nM Nucleic acid template 50 copies Ultra-pure water Make up to 25 μL

[0088] Among them, the 2X PCR reaction buffer at least includes: 3 mmol / L of MgCl2, 30 mmol / L of Tris-HCl with a pH of 8.3, 0.5 mmol / L of dNTP, and 70 mmol / L of (NH4)2SO4.

[0089] Example 1

[0090] Primer-probe combination for detecting EVA71

[0091] The forward primer is as shown in SEQ ID NO:1, the reverse primer is as shown in SEQ ID NO:2, and the detection probe is as described in SEQ ID NO:3. The 15 bases at the 5' end of the forward primer are reverse complementary to the 15 bases at the 5' end of the detection probe. The sequences of the primer-probe combination are shown in Table 2.

[0092] Table 2

[0093]

[0094] Example 2

[0095] Detection of Enterovirus 71 (EVA71)

[0096] Prepare according to the reaction system shown in Table 1 and the primer-probe combination shown in Table 2, and then perform PCR reaction according to the reaction program in Table 3. The nucleic acid template is Enterovirus 71 (EVA71) plasmid. After quantification and dilution, it is mixed with the reaction system at 50 copies / reaction. And adopt digital PCR photography method or melting curve analysis of qPCR at 40-85 °C, with a heating rate of 0.05 °C per second for light collection.

[0097] Table 3

[0098]

[0099] Compared with EVA71, there are only 2-3 base differences in the conserved regions of the designed probes for types 6 (CA6) and 16 (CA16) of group A Coxsackievirus. Use these two plasmids as controls for digital PCR reaction, and verify whether the two plasmids can be distinguished by the primer-probe designed by the technical scheme of the present invention, where NTC is pure water. The specific results are as Figure 1 shown. When performing digital PCR detection, constant temperature photography is carried out at 40 °C. Because there are 2-3 base types and quantity differences in the detection regions of the probe and different plasmids, the fluorescence signals are different accordingly. Therefore, the technical scheme of the present invention can distinguish interference sequences with only 2-3 base differences in the detection region through the difference in fluorescence signals.

[0100] Furthermore, as Figure 2 shown, after qPCR reaction and melting curve analysis, it can be seen that the melting curve peak of EVA71 (57.9 °C) has a difference greater than 5 °C from the peaks of CA6 (51.9 °C) or CA16. Therefore, the nucleic acid sequences with single-digit base differences can be well distinguished by the technical scheme of the present invention.

[0101] Example 3

[0102] Effect of the concentration ratio of forward primer to reverse primer on detection

[0103] Prepare according to the reaction system in Table 1, and only adjust the concentration ratio of the forward primer to the reverse primer appropriately as shown in Table 4. The reaction program is the same as that in Example 2. The results are as Figure 3 shown. When the concentration ratio of the forward primer to the reverse primer is different, the amplification efficiency is also different. The ratio of the forward and reverse primers has a great influence on the fluorescence value of the positive droplets. When the ratio of the forward primer to the reverse primer is about 6:2-6:1, the effect is the best.

[0104] Table 4

[0105] Number Forward primer SEQ ID NO:1 Reverse primer SEQ ID NO:1 1 500 nM 500 nM 2 500 nM 320 nM 3 500 nM 160 nM 4 500 nM 80 nM 5 320 nM 500 nM 6 160 nM 500 nM 7 80 nM 500 nM

[0106] Example 4

[0107] Effect of the length of the complementary sequence (anchoring binding region) between the detection probe and the forward primer on detection

[0108] The primer-probe combinations are shown in Table 5. The primer-probe combination a in Example 1 has a 15-bp base complementarity between the detection probe and the forward primer. The detection probe of primer-probe combination b has a 13-bp base complementarity with the forward primer; the detection probe of primer-probe combination c has an 11-bp base complementarity with the forward primer; the detection probe of primer-probe combination d has a 9-bp base complementarity with the forward primer.

[0109] Table 5

[0110]

[0111]

[0112] As Figure 4 shown, after digital PCR reaction and taking pictures at a constant temperature at different temperatures, it can be seen that the longer the sequence complementary between the detection probe and the forward primer, the more stable its positive signal is under temperature changes. It also further shows that the complementarity between the detection probe and the forward primer can help the detection probe bind to the target sequence.

[0113] Example 5

[0114] Effect of the length of the detection region of the detection probe on detection

[0115] The forward primer (SEQ ID NO:1) and reverse primer (SEQ ID NO:2) in Example 1 and a control primer were used. The control primer is a forward primer that does not hybridize with the detection probe. The hybridization length between the detection probe and the forward primer is the same, and the lengths of the detection regions are different, as shown in Table 6 specifically.

[0116] Table 6

[0117]

[0118] As Figure 5 、 6As shown, the longer the detection region of the detection probe, the easier it is for the detection probe to bind to the target sequence and generate a detectable signal. When the forward primer contains an anchoring region complementary to the detection probe, it is easier to help the detection probe bind to the target sequence to generate a detectable signal. Moreover, it can be seen from taking pictures at different temperatures that using the technical solution of the present invention is more conducive to the detection probe binding to the target and strand separation, and thus is conducive to achieving multiplex detection at multiple temperatures using the technical solution of the present invention, reducing the temperature requirements for multiplex detection.

[0119] Example 6

[0120] Relationship between the anchoring binding region of the detection probe and the primer anchoring region

[0121] By designing the probe anchoring binding region and the primer anchoring region to be complementary or identical, and the specific sequences are shown in Table 7, understand the influence of the relationship between the probe anchoring binding region and the primer anchoring region on the detectable signal value.

[0122] Table 7

[0123]

[0124] From Figure 7 it can be seen that compared with the forward primer without an anchoring region, the forward primer with an anchoring region can help the detection probe hybridize to the target sequence. The detectable signal of the detection probe anchoring binding region complementary to the primer is better than that of the detection probe anchoring binding region identical to the primer.

[0125] Example 7

[0126] Primer-probe combination for detecting EVA71 - the detection region of the detection probe is set at the 5' end

[0127] The forward primer is as shown in SEQ ID NO:13, the reverse primer is as shown in SEQ ID NO:14, the detection probe is as described in SEQ ID NO:15, the 15 bases at the 5' end of the forward primer are reverse complementary to the 15 bases at the 3' end of the detection probe, and the sequences of the primer-probe combination are shown in Table 2.

[0128] Table 8

[0129]

[0130] From Figure 8It can be seen that when the detection region of the detection probe is at the 5' end, the amplification efficiency of the anchoring binding region complementary to the primer is the best. For primers with the same non-extension group and the same extension group and the same anchoring binding region of the detection probe, in the same non-extension group, since multiple A's are designed at the 5' end of the primer, it can be avoided that the primer hybridizes with the detection probe after being replicated and continues to amplify using the detection probe as a template. Therefore, it can be considered that although there are more positive signals in the same extension group than in the same non-extension group, it is very likely that false positive signals are affecting. In summary, when the detection region of the detection probe is set at the 5' end, the maximum value of positive signals that can be detected by complementarity with the anchoring region of the primer is obtained.

[0131] Example 8

[0132] The fluorescent group and the quenching group are respectively set at the 5' end and the 3' end of the detection probe; if the detection probe hybridizes with the primer, the background of the negative droplets is extremely high, and basically no positive droplets are detected.

[0133] Example 9

[0134] The functional coding region of the BRAF gene consists of 2,510 base pairs, located on human chromosome 7q34, encoding a serine / threonine protein kinase in the MAPK pathway. This enzyme transduces signals from RAS to MEK1 / 2, thus participating in cell regulation. BRAF gene mutations have been found in a variety of malignant tumor cells. These mutations mainly occur in exon 15 encoding the activation region, and about 92% of them are located at nucleotide position 1799 (1799T>A), resulting in the substitution of valine encoded by it with glutamic acid (V600E). In addition to lung cancer, BRAF mutations exist in different proportions in colon cancer, melanoma, thyroid cancer, liver cancer, pancreatic cancer, etc.

[0135] There are relatively many mutations at the V600 position of the BRAF gene, and there is only a difference of 1 base between some mutations. The common 3 types of base mutations are: 1798_1799GT>AG, 1797_1799AGT>GAG, 1798_1799GT>CG. It can be seen that 1798_1799GT>AG has only a difference of 1 base from the other two mutation types; in addition, there is also only a difference of 1 base between BRAF V600K 1798_1799GT>AA and 1798_1799GT>AG. Therefore, it is very difficult to ensure the specificity of the detection of the probe designed for this detection target. The primer-probe combinations designed using the technical solution of the present invention are shown in the following table.

[0136] Table 10

[0137]

[0138] Using different detection samples (Sample 1 - BRAF V600 WT, Sample 2 - BRAF V600K 1798_1799GT>AA, Sample 3 - 1797_1799AGT>GAG, Sample 4 - 1798_1799GT>CG, Sample 5 - 1798_1799GT>AG), digital PCR reactions (without reverse transcription process) were carried out using the reaction system in Table 1 and the reaction procedure in Table 2, and photographs were taken at different temperatures. From Figure 9 It can be seen that using the scheme design of the present invention, only weak non-specificity appears for BRAF V600K 1798_1799GT>AA at 30°C, and obvious non-specific amplification appears for 1797_1799AGT>GAG; however, when the photographing temperature is increased to 40°C, except for the specific amplification of 1798_1799GT>AG, no non-specific amplification appears for the remaining 4 templates. Therefore, by taking photographs at different temperatures, the targets with individual base differences can be distinguished, further improving the specificity of detection.

Claims

1. A primer-probe combination for nucleic acid detection by PCR, characterized in that The primer-probe combination includes a first oligonucleotide, a second oligonucleotide, and a detection probe. The first oligonucleotide and the second oligonucleotide are a forward primer and a reverse primer to each other. The first oligonucleotide includes an anchoring region that is not complementary or identical to the target sequence, and a binding region that is complementary to the target sequence. The detection probe includes an anchoring binding region that is complementary or identical to the anchoring region of the first oligonucleotide, and a detection region that is complementary to the target sequence; the detection probe is labeled with a detection group.

2. The primer-probe combination according to claim 1, characterized in that The anchoring region of the first oligonucleotide is upstream of the binding region.

3. The primer-probe combination according to claim 1, characterized in that The anchoring binding region of the detection probe has a length of 9 to 16 bases, and the detection region of the detection probe has a length of 9 to 16 bases. Preferably, the 3'-end of the detection probe is blocked; more preferably, the 3'-end blocking is selected from one of amino group blocking modification, C3-Spacer modification, thiol modification, dideoxy modification, phosphate group modification, and detection group modification.

4. The primer-probe combination according to claim 1, characterized in that The detection group includes a fluorescent group and a quenching group.

5. A reagent for nucleic acid detection by PCR, characterized in that, The reagent includes the primer-probe combination according to any one of claims 1 to 4; preferably, the reagent further includes dNTP, buffer, salt ions, and polymerase.

6. A kit for nucleic acid detection by PCR, characterized in that, The kit includes the primer-probe combination according to any one of claims 1 to 4, or the reagent according to claim 5.

7. A method for specifically detecting target nucleic acids with single-digit base differences, characterized in that Comprising the following steps: a) Contacting a sample containing a target nucleic acid with the first oligonucleotide, the second oligonucleotide, and the detection probe; The first oligonucleotide and the second oligonucleotide are a forward primer and a reverse primer to each other. The first oligonucleotide includes an anchoring region that is not complementary or identical to the target sequence, and a binding region that is complementary to the target sequence. The detection probe includes an anchoring binding region that is complementary or identical to the anchoring region of the first oligonucleotide, and a detection region that is complementary to the target sequence; the detection region is labeled with a detection group; b) Performing a PCR reaction on the mixture in step a) to form an amplification product; c) Specifically detecting the target nucleic acid with single-digit base differences based on the melting analysis or hybridization analysis of the amplification product.

8. The detection method according to claim 7, characterized in that It further includes a step of performing a reverse transcription reaction using a reverse transcriptase to form a reverse transcription product.

9. The detection method according to claim 7, characterized in that When the anchoring binding region of the detection probe is complementary to the anchoring region of the first oligonucleotide, the concentration of the first oligonucleotide is higher than that of the second oligonucleotide; the concentration ratio of the first oligonucleotide to the second oligonucleotide is 6:6 to 6:1, preferably the concentration ratio is 6:2 to 6:

1.

10. The detection method according to claim 7, characterized in that When the anchoring binding region of the detection probe is identical to the anchoring region of the first oligonucleotide, the concentration of the first oligonucleotide is lower than that of the second oligonucleotide; the concentration ratio of the first oligonucleotide to the second oligonucleotide is 1:6 to 6:6; preferably the concentration ratio is 1:6 to 2:6.

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