CN113186252B - Double-stranded nucleic acid specificity detection probe at normal temperature and corresponding detection method - Google Patents
Double-stranded nucleic acid specificity detection probe at normal temperature and corresponding detection method Download PDFInfo
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- CN113186252B CN113186252B CN202110313792.8A CN202110313792A CN113186252B CN 113186252 B CN113186252 B CN 113186252B CN 202110313792 A CN202110313792 A CN 202110313792A CN 113186252 B CN113186252 B CN 113186252B
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Abstract
The invention discloses a double-stranded nucleic acid specificity detection probe at normal temperature and a detection method using the probe. The detection method provided by the invention has the advantages that the lambda exo is used for driving the target double-stranded nucleic acid to perform a strand displacement reaction with the designed probe, the probe is digested after the optimal substrate of the enzyme is formed to generate a detection signal, the lambda exo identifies and digests the 5' phosphate end of the nucleic acid to initiate the digestion reaction of the enzyme and generate a fluorescent signal, the signal strictly corresponds to the target nucleic acid sequence, the target sequence and the non-target sequence are distinguished with high reliability, the double-stranded nucleic acid target can be directly detected at room temperature, the detection program of the double-stranded nucleic acid target object is greatly simplified, the in-situ application difficulty of the double-stranded nucleic acid target object is reduced, and the method can be widely applied to nucleic acid detection molecule diagnosis.
Description
Technical Field
The invention relates to a double-stranded nucleic acid specificity detection probe at normal temperature and a corresponding detection method in the field of biotechnology.
Background
Nucleic acid molecule identification and detection are very important for prevention, control, diagnosis and treatment of diseases such as tumor, virus infection and the like, the existing nucleic acid detection technology is mostly based on PCR amplification at present, and because strict temperature gradient control is required, the time is consumed, and an accurate instrument is required to control the experiment temperature. The establishment of a reliable and accurate nucleic acid detection method at normal temperature is of great significance for in vitro diagnosis. Some current isothermal amplification methods, such as RPA, LAMP, can amplify nucleic acids efficiently at normal temperature, but specific signal output and detection are difficult to achieve using only these methods. Most of the genomes of biological samples are double-stranded nucleic acids, and the products of isothermal amplification are also double-stranded nucleic acids. The detection of double-stranded nucleic acid in nucleic acid molecular diagnosis needs special primer design, enzyme digestion and other pretreatment steps, and the genome nucleic acid fragment can enter a detection system to be effectively detected after being digested into a single-stranded oligonucleotide fragment, so that the efficiency of nucleic acid molecular detection is greatly limited. In combination with the above factors, it is important to perform specific recognition of a double-stranded nucleic acid at room temperature. The existing technologies include nucleic acid detection and gene editing technology (CRISPR) with multiple enzymes. The multi-enzyme combination method is mainly used for detecting double-stranded nucleic acid by comprehensively utilizing the functions of recombinase, helicase and the like for unwinding double strands at normal temperature and the substrate recognition and degradation functions of some nucleases (endonuclease IV, APE1, exoIII and the like). The use of multiple enzymes increases the complexity and uncertainty of the detection. Furthermore, the compatibility of multiple enzymes is problematic. In a detection technology based on gene editing, a Cas protein specifically recognizes a specific nucleic acid sequence under the guidance of gRNA/DNA at room temperature, but recognition of the Cas protein depends on a PAM sequence, and a target miss phenomenon is serious. In conclusion, the new method for detecting the double-stranded nucleic acid at the normal temperature (room temperature) is expected to break through the bottleneck of the detection of the nucleic acid at the normal temperature.
Disclosure of Invention
The technical problem to be solved by the invention is how to detect the double-stranded nucleic acid at room temperature.
In order to solve the technical problems, the invention firstly provides a double-stranded nucleic acid specificity detection probe at normal temperature.
The invention provides a double-stranded nucleic acid specificity detection probe at normal temperature, which comprises a fluorescent probe, a quenching probe and single-stranded DNA (deoxyribonucleic acid) containing a target probe binding domain; the fluorescent probe is marked with a fluorescent group, and the quenching probe is marked with a quenching group; the target probe binding domain is stably combined with one strand of the double-stranded DNA target; the fluorescent probe and the quenching probe are connected or combined together through single-stranded DNA (deoxyribonucleic acid) containing a target probe binding domain, the quenching group can quench a fluorescent signal of the fluorescent group, and the 5' end of the single-stranded DNA containing the target probe binding domain is subjected to phosphorylation modification; all the 5' ends of the single-stranded DNA which is not modified by other strands in the double-stranded nucleic acid specific detection probe at the normal temperature are modified by sulfo.
In the double-stranded nucleic acid specific detection probe at normal temperature, the thio modification is carried out to modify 4-5 nucleotides, and specifically, the thio modification can be carried out to modify 5 nucleotides.
In the double-stranded nucleic acid specific detection probe at normal temperature, the quenching group can be BHQ1; the fluorophore may optionally be FAM.
The double-stranded nucleic acid specific detection probe at normal temperature may specifically be any one of a, B, and C:
a single-stranded DNA type probe;
b type composite probe;
c, two kinds of composite probes.
In the double-stranded nucleic acid specific detection probe at normal temperature, the single-stranded DNA type probe A is a single-stranded DNA with a target probe binding domain, and the single-stranded DNA is marked with a fluorescent group and a quenching group; the fluorescent signal of the fluorophore can be quenched by the quencher.
In the double-stranded nucleic acid specific detection probe at normal temperature, the 5' end of the A is modified with a phosphate group; the quenching group in A is modified at the 3' end; the fluorescent group is modified on the T base.
In the double-stranded nucleic acid specific detection probe at normal temperature, the composite probe B is I type or II type:
type I consists of two single-stranded DNAs B1-I and B2-I:
B1-I quenching probe;
the B2-I fluorescent group marked recognition probe is formed by connecting a target binding domain and a quenching probe complementary domain in sequence; wherein the quenching probe complementary domain stably binds to the B1-I quenching probe;
type II consists of two single-stranded DNAs B1-II and B2-II:
B1-II fluorescent probes;
the B2-II quenching group marked identification probe is formed by sequentially connecting a target binding domain and a fluorescent probe complementary domain; wherein the fluorescent probe complementary domain is stably combined with the B1-II fluorescent probe.
Wherein, the 5 'end of the B2-I is modified with phosphate group, and the fluorescent group of the B2-I is modified on the T base close to the 3' end; the quenching group of the B1-I quenching probe is modified at the 3 'end, the 5' end of the B1-I quenching probe is modified by sulfo, and the length of the sulfo modification is 4-5 nucleotides.
Wherein, the 5 'end of the B2-II is modified with phosphate group, and the quenching group of the B2-II is modified at the 3' end; the fluorescent group of the B1-II fluorescent probe is modified on a T basic group close to the 3 'end, the 5' end of the B1-II is subjected to sulfo modification, and the length of the sulfo modification is 4-5 nucleotides.
Wherein the target binding domain is 45 nucleotides in length.
Wherein the molar ratio of B1-I to B2-I is 1; the molar ratio of B1-II to B2-II is 1.
In the double-stranded nucleic acid specific detection probe at normal temperature, the second type of composite probe C consists of three DNA single strands of C1-C3:
a C1 fluorescent probe;
a C2 quenching probe;
a C3 recognition probe, namely single-stranded DNA (deoxyribonucleic acid) which is formed by sequentially connecting a target probe binding domain, a fluorescent probe complementary domain and a quenching probe complementary domain according to the sequence from 5 'end to 3' end;
the C1 is stably combined with the complementary domain of the fluorescent probe in the C3; the C2 stably binds to the quenching probe complementary domain in the C3.
Wherein, the fluorescent group in C1 is modified on the T base close to the 5' end; the quenching group in the C2 is modified at the 3 'end, the 5' end of the C2 is subjected to thio modification, and the length of the thio modification is 4-5 nucleotides; the 5' end of the C3 is modified with a phosphate group.
Wherein, the molar ratio of C1, C2 and C3 is 1.
Wherein, the length of the C1 is 20-25 nucleotides; the length of C2 is 20-25 nucleotides; c3 Wherein the target probe-binding domain is 45 nucleotides in length.
Among the above-mentioned double-stranded nucleic acid specific detection probes at normal temperature, the double-stranded nucleic acid specific detection probes at normal temperature may be any one of W1 to W6:
w1, using a double chain formed by a sequence 1 and a sequence 2 in a sequence table as a target object, or using a double chain formed by a sequence 3 and a sequence 4 in the sequence table as a target object, wherein the double-stranded nucleic acid specific detection probe at normal temperature is a single-stranded DNA shown in a sequence 5 in the sequence table;
w2, taking a double chain formed by a sequence 1 and a sequence 2 in a sequence table as a target object, or taking a double chain formed by a sequence 3 and a sequence 4 in the sequence table as a target object, wherein the double-stranded nucleic acid specificity detection probe at normal temperature is composed of a single-stranded DNA shown by a sequence 6 in the sequence table and a single-stranded DNA shown by a sequence 7 in the sequence table;
w3, taking a double chain formed by a sequence 1 and a sequence 2 in a sequence table as a target object, or taking a double chain formed by a sequence 3 and a sequence 4 in the sequence table as a target object, wherein the double-stranded nucleic acid specificity detection probe at normal temperature is composed of a single-stranded DNA shown by a sequence 8 in the sequence table and a single-stranded DNA shown by a sequence 9 in the sequence table;
w4, a double strand consisting of a sequence 1 and a sequence 2 in a sequence table is taken as a target object, or a double strand consisting of a sequence 3 and a sequence 4 in the sequence table is taken as a target object, and the double-stranded nucleic acid specific detection probe at normal temperature consists of a single-stranded DNA shown in a sequence 10 in the sequence table, a single-stranded DNA shown in a sequence 11 in the sequence table, and a single-stranded DNA shown in a sequence 12 in the sequence table;
w5, taking a double strand consisting of a sequence 13 and a sequence 14 in a sequence table as a target object, wherein the double-strand nucleic acid specific detection probe at normal temperature consists of a single-strand DNA shown in a sequence 10 in the sequence table, a single-strand DNA shown in a sequence 11 in the sequence table and a single-strand DNA shown in a sequence 15 in the sequence table;
w6, a double strand consisting of a sequence 16 and a sequence 17 in a sequence table is taken as a target object, and the double-stranded nucleic acid specific detection probe at the normal temperature is composed of a single-stranded DNA shown in a sequence 10 in the sequence table, a single-stranded DNA shown in a sequence 11 in the sequence table, and a single-stranded DNA shown in a sequence 18 in the sequence table.
The invention also provides a nucleic acid detection method using the double-stranded nucleic acid specificity detection probe at normal temperature, which comprises the steps of mixing the double-stranded nucleic acid specificity detection probe at normal temperature with a sample to be detected, lambda phage exonuclease (lambda exo) and single-stranded binding protein (SSB), detecting a fluorescent signal, and judging whether the sample to be detected contains a target object, wherein the judgment standard is as follows: if a double-stranded DNA target exists in the sample to be detected, the fluorescence value is obviously increased; and if the target does not exist in the sample to be detected, the fluorescence value does not have obvious rising change.
The invention also provides a product consisting of X1 and X2; the X1 is the double-stranded nucleic acid specific detection probe at normal temperature, and the X2 is a reagent and/or an instrument required for detection.
In the product, the reagent required for detection can contain lambda phage exonuclease, single-stranded binding protein and reaction buffer solution; the apparatus may be a nutTime fluorescence detector. The reaction buffer may be formulated with 67mM Glycine-KOH,2.5mM MgCl 2 ,50mM KCl,50mM NH 4 Cl,50μg/ml BSA,pH 9.4。
In order to solve the technical problems, the invention also provides the double-stranded nucleic acid specificity detection probe at normal temperature, the method and the application of the product in preparing a nucleic acid detection reagent.
In the present invention, the inventors have constructed a probe system for detecting double-stranded nucleic acid at normal temperature, using the special properties of lambda phage exonuclease (. Lamda. Exo): the lambda exo is used for driving target double-stranded nucleic acid to perform a strand displacement reaction with a designed probe, after an optimal substrate of an enzyme is formed, the probe is digested to generate a detection signal, the lambda exo recognizes and digests the 5' phosphate end of the nucleic acid to initiate the digestion reaction of the enzyme, and a fluorescent signal is generated, wherein the signal strictly corresponds to a target nucleic acid sequence. The detection method can directly detect the double-stranded nucleic acid target at the temperature of 25-37 ℃, can distinguish a target sequence from a non-target sequence with high reliability, can greatly simplify the detection procedure of the double-stranded nucleic acid target, reduces the difficulty of the on-site application of the double-stranded nucleic acid target, and can be widely applied to molecular diagnosis based on nucleic acid detection. The invention can combine polymerase-recombinase amplification (RPA), loop-mediated isothermal amplification (LAMP) and the like to realize high-sensitivity and high-specificity nucleic acid detection at normal temperature. The invention is also expected to be used for in-situ specific recognition and imaging of double-stranded nucleic acid in living cells.
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FIG. 1 is a schematic diagram showing a detection reaction of the single-stranded DNA type probe of the present invention.
FIG. 2 is a graph showing fluorescence-time traces of detection of a double-stranded target by a single-stranded DNA type probe in example 1 of the present invention. Wherein, the graph A in FIG. 2 is for detecting a target (double strand consisting of T1-1 and T1-2) having a length equal to that of the probe recognition region, and the graph B in FIG. 2 is for detecting a target (double strand consisting of T2-1 and T2-2) having a length longer than that of the probe recognition region.
FIG. 3 is a schematic diagram of a detection reaction of a class of composite probes of the present invention.
FIG. 4 is a fluorescent-time trace diagram of a type of composite probe for detecting a double-stranded target in example 2 of the present invention. Wherein, the graph A in FIG. 4 is for detecting a target (double strand consisting of T1-1 and T1-2) having a length equal to that of the probe recognition region, and the graph B in FIG. 4 is for detecting a target (double strand consisting of T2-1 and T2-2) having a length longer than that of the probe recognition region.
FIG. 5 is a schematic diagram of the detection reaction of the second type of composite probe of the present invention.
FIG. 6 shows fluorescence-time traces and gel electrophoresis characterization of two types of composite probes in example 3 of the present invention for detecting a target (a double-strand consisting of T1-1 and T1-2) with a length equal to the probe recognition region. FIG. 6A is a graph showing a fluorescence-time trace using a double-stranded nucleic acid-specific detection probe W4; panel B of fig. 6 is a characterization of gel electrophoresis, where M:25bp marker,1: single-stranded target sequence, 2: double-stranded target sequence, 3: double-stranded target sequence + λ exo,4: universal discrimination probe W4,5: universal recognition probe + λ exo,6: double-stranded target sequence + universal recognition probe, 7: double-stranded target sequence + universal recognition probe + λ exo,8: single-stranded target sequence + universal recognition probe, 9: single-stranded target sequence + universal recognition probe + λ exo,10: a fluorescent probe (C1) and a quenching probe (C2).
FIG. 7 shows fluorescence-time traces and gel electrophoresis characterization of two types of composite probes in example 3 of the present invention for detecting a target (double-stranded T2-1 and T2-2) longer than the probe recognition region. FIG. 7A is a graph A showing a fluorescence-time trace using a double-stranded nucleic acid-specific detection probe W4; panel B of FIG. 7 is a representation of gel electrophoresis, where M:25bp marker,1: single-stranded target sequence, 2: double-stranded target sequence, 3: double-stranded target sequence + λ exo,4: universal discrimination probe W4,5: universal recognition probe + λ exo,6: double-stranded target sequence + universal recognition probe, 7: double-stranded target sequence + universal recognition probe + λ exo,8: single-stranded target sequence + universal recognition probe, 9: single-stranded target sequence + universal recognition probe + λ exo,10: a fluorescent probe (C1) and a quenching probe (C2).
FIG. 8 is a fluorescent-time trace diagram of two types of composite probes for detecting double-stranded target in example 4 of the present invention. Wherein, the A picture of figure 8 is used for detecting the E gene segment in SARV-CoV-2, and the used probe is W6; FIG. 8B is a diagram showing the detection of the N gene fragment in SARV-CoV-2 using W5 as a probe.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
In the quantitative tests in the following examples, three replicates were set up and the results averaged.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The detection method is an identification method for detecting a double-stranded nucleic acid target object by using a DNA probe and lambda phage exonuclease (lambda exo), the used double-stranded nucleic acid specificity detection probe at normal temperature is provided with a fluorescent group and a quenching group, is complementary with one single strand of the target object and can perform strand displacement reaction at normal temperature, and the displaced probe is destructured to form an optimal substrate of the exonuclease (lambda exo) and then is digested to generate a detection signal. Specifically, the method comprises the following steps:
s1, designing a double-stranded nucleic acid specific detection probe at normal temperature according to a double-stranded DNA target object, wherein the double-stranded nucleic acid specific detection probe at normal temperature comprises a fluorescent probe, a quenching probe and single-stranded DNA containing a target probe binding domain; the fluorescent probe is marked with a fluorescent group, and the quenching probe is marked with a quenching group; the target probe binding domain is stably combined with one strand of the double-stranded DNA target; the fluorescent probe and the quenching probe are connected or combined together through single-stranded DNA (deoxyribonucleic acid) containing a target probe binding domain, the quenching group can quench a fluorescent signal of the fluorescent group, and the 5' end of the single-stranded DNA containing the target probe binding domain is subjected to phosphorylation modification; all single-stranded DNA in the double-stranded nucleic acid specific detection probe at normal temperature is subjected to sulfo-modification at the 5' end without other modifications.
The double-stranded nucleic acid specific detection probe at normal temperature can be any one of a single-stranded DNA type probe (see example 1, the flow is shown in figure 1), a first type of composite probe (see example 2, the flow is shown in figure 3) and a second type of composite probe (see example 3, example 4 and the flow is shown in figure 5).
S2, adding a sample to be detected, exonuclease (lambda exo) and single-stranded binding protein (SSB) into a probe system containing the double-stranded nucleic acid specificity detection probe in the S1 at normal temperature to form a detection system, and recording fluorescence intensity;
s3, analyzing the fluorescence intensity of the detection record to determine whether a double-stranded DNA target exists in the sample to be detected:
if a double-stranded DNA target exists in a sample to be detected, after the double-stranded DNA target and a double-stranded nucleic acid specificity detection probe are subjected to strand displacement reaction at normal temperature, the double-stranded nucleic acid specificity detection probe is destructured at normal temperature to form an optimal digestion substrate of exonuclease (lambda exo), a high-resolution fluorescence signal is generated after digestion of the exonuclease (lambda exo), and the fluorescence value is obviously increased;
if the target does not exist in the sample to be detected, the fluorescence value does not have obvious rising change.
The reaction buffer environment 1 X.lamda.exo buffer in the following examples is a product of New England Biolabs (NEB) under the trade name M0262S, and the reagent contains exonuclease (. Lamda.exo).
Example 1 detection method Using Single-stranded DNA-type Probe
In this embodiment, a double-stranded DNA target is detected based on a single-stranded DNA type probe, specifically, the double-stranded DNA target is a double strand composed of T1-1 and T1-2 (a target having a length equal to a probe recognition region) or a double strand composed of T2-1 and T2-2 (a target having a length longer than the probe recognition region), the sample to be detected is the artificially synthesized double strand, and a blank control is set. The method comprises the following specific steps:
s1, designing a double-stranded nucleic acid specific detection probe at normal temperature according to a double-stranded DNA target, wherein the double-stranded nucleic acid specific detection probe at normal temperature used in the embodiment is a single-stranded DNA type probe, and the specific sequence is shown as A in Table 1:
TABLE 1 nucleic acid sequences
Note: in Table 1, P represents phosphorylation.
FIG. 1 is a schematic diagram of a single-stranded DNA probe designed to have the following DNA single strands (see A in Table 1):
a is a single DNA strand (which can be regarded as having the function of a recognition probe and can be 45 nucleotides in length) with a target probe binding domain, and the single DNA strand is marked with a fluorescent group (which can be regarded as having the function of a fluorescent probe) and a quenching group (which can be regarded as having the function of a quenching probe); the fluorescent signal of the fluorophore can be quenched by the quencher.
Wherein, the 5' end of A is modified with a phosphate group; the quenching group in the A is modified at the 3' end, and the quenching group is selected as BHQ1; the fluorescent group is modified on the T base, and is selected as FAM.
Namely, the following set of specific detection probes W1 for the double-stranded nucleic acid at normal temperature is designed and obtained:
w1, double chains formed by a sequence 1 and a sequence 2 in a sequence table are used as a target object, or double chains formed by a sequence 3 and a sequence 4 in the sequence table are used as the target object, and the double-stranded nucleic acid specific detection probe at normal temperature is single-stranded DNA shown in a sequence 5 in the sequence table.
The desired double-stranded nucleic acid-specific detection probe W1 at room temperature was synthesized as shown in Table 1.
S2, adding a sample to be detected, exonuclease (lambda exo) and single-stranded binding protein (SSB) into a probe system containing the double-stranded nucleic acid specificity detection probe in the S1 at normal temperature to form a detection system, and recording fluorescence intensity;
s2-1 establishment of Probe System
Set up probe system (20 μ L): adding a double-stranded nucleic acid specific detection probe at normal temperature into a reaction buffer solution with the environment of 1 x lambda exobaufer, wherein the double-stranded nucleic acid specific detection probe at normal temperature is W1, and the concentration of single-stranded DNA in the double-stranded nucleic acid specific detection probe at normal temperature in a system is 1 mu M. Make the total volume of the system equal to 20. Mu.L, the rest volume is ddH 2 And (4) supplementing by using oxygen.
A probe system 1 is established by using a double-stranded nucleic acid specific detection probe W1 at normal temperature.
S2-2 detection
20 μ L assay system: mu.L of the above probe system (probe system 1) having a concentration of 1. Mu.M was taken so that the concentration thereof in the system became 100nM, 10. Mu.L of the sample to be tested was added, lambda exo was added so that the concentration thereof in the system became 50U/mL, and SSB was added so that the concentration thereof in the system became 500nM. If the total volume of the system is less than 20 mu L, ddH is used 2 And (4) supplementing by using oxygen. And (3) putting the sample into a real-time fluorescence detector for detection at 37 ℃, setting the detection time interval to be 10 seconds for one cycle, setting the cycle number to be 180 times, and setting the detection time to be 30 minutes to obtain the fluorescence intensity-time track. When the target nucleic acid sequence does not exist in the detection target, the fluorescence value has no obvious ascending change; if the fluorescence value increases significantly, the target nucleic acid sequence is considered to be present in the detection target.
S3, analyzing the fluorescence intensity recorded in the detection to determine whether a double-stranded DNA target exists in the sample to be detected:
the fluorescence-time trace when a target (double strand consisting of T1-1 and T1-2) of equal length to the probe recognition region was detected is shown in panel A of FIG. 2. The fluorescence-time trace when a target longer than the probe recognition region (double strand consisting of T2-1 and T2-2) was detected is shown in panel B of FIG. 2. The result shows that if a double-stranded DNA target object exists in the sample to be detected, the fluorescence value of the target object is obviously increased no matter the target object is the target object with the same length as the probe identification area or the target object is the target object with the length longer than the probe identification area; and if the target does not exist in the sample to be detected, the fluorescence value does not have obvious rising change.
The result shows that the single-stranded DNA type probe can effectively detect the double-stranded target object.
Example 2 detection method Using one type of composite Probe
In this embodiment, a double-stranded DNA target is detected based on a kind of composite probe, specifically, the double-stranded DNA target is a double strand composed of T1-1 and T1-2 (a target having a length equal to a probe recognition region) or a double strand composed of T2-1 and T2-2 (a target having a length longer than the probe recognition region), the sample to be detected is the artificially synthesized double strand, and a blank control is set. The method comprises the following specific steps:
s1, designing a double-stranded nucleic acid specific detection probe at normal temperature according to a double-stranded DNA target, wherein the double-stranded nucleic acid specific detection probe at normal temperature used in the embodiment is a composite probe, and the specific sequence is shown in Table 2:
TABLE 2 nucleic acid sequences
Note: in table 2P represents phosphorylation, thio, underlined is the quenching probe complementary domain, double underlined is the fluorescent probe complementary domain.
FIG. 3 is a schematic structural diagram of a type of composite probe, wherein the type of composite probe is designed to be type I or type II:
type I consists of two single-stranded DNAs B1-I and B2-I:
B1-I quenching probe (single-stranded DNA with quenching group mark, namely quenching signal probe, the length can be 20-25 nucleotides);
the B2-I fluorescent group labeled recognition probe is formed by sequentially connecting a target binding domain (namely the target probe binding domain in figure 3) and a quenching probe complementary domain; wherein the quenching probe complementary domain stably binds to the B1-I quenching probe (the quenching probe complementary domain is underlined and the target binding domain is the non-underlined portion in B2-I of Table 2);
the type I probe satisfies that the fluorescent signal of the fluorescent group of B2-I can be quenched by the quenching group of B1-I.
Wherein, the 5 'end of the B2-I is modified with a phosphate group, the fluorescent group of the B2-I is modified on a T base close to the 3' end, and the fluorescent group is selected as FAM; the quenching group of the B1-I quenching probe is modified at the 3 'end, the quenching group is BHQ1, the 5' end of the B1-I quenching probe is subjected to sulfo-modification, and the length of the sulfo-modification is 4-5 nucleotides.
The target binding domain is 45 nucleotides in length.
The molar ratio of B1-I to B2-I is 1.
Type II consists of two single-stranded DNAs B1-II and B2-II:
B1-II fluorescent probes (single-stranded DNA labeled with a fluorophore, i.e., the fluorescent signaling probe in FIG. 3, which can be 20-25 nucleotides in length);
the B2-II quenching group labeled recognition probe is formed by sequentially connecting a target binding domain (namely the target probe binding domain in figure 3) and a fluorescent probe complementary domain; wherein the fluorescent probe complementary domain stably binds to the B1-II fluorescent probe (underlined in B2-II of Table 2 are fluorescent probe complementary domains, and the non-underlined part is a target binding domain);
the II type probe satisfies that the fluorescence signal of the fluorescent group of B1-II can be quenched by the quenching group of B2-II.
Wherein, the 5 'end of the B2-II is modified with phosphate group, the quenching group of the B2-II is modified at the 3' end, and the quenching group is selected as BHQ1; the fluorescent group of the B1-II fluorescent probe is modified on a T base close to the 3 'end, the fluorescent group is selected as FAM, the 5' end of the B1-II is subjected to sulfo modification, and the length of the sulfo modification is 4-5 nucleotides. The target binding domain is 45 nucleotides in length.
The molar ratio of B1-II to B2-II is 1.
Namely, the following two sets of double-stranded nucleic acid specific detection probes W2 and W3 (see Table 2) at normal temperature are obtained by design:
w2, double chains formed by a sequence 1 and a sequence 2 in a sequence table are used as a target object, or double chains formed by a sequence 3 and a sequence 4 in the sequence table are used as a target object, and the double-stranded nucleic acid specificity detection probe at the normal temperature is composed of single-stranded DNA shown in a sequence 6 in the sequence table and single-stranded DNA shown in a sequence 7 in the sequence table (I type probe).
W3, taking a double chain formed by a sequence 1 and a sequence 2 in a sequence table as a target object, or taking a double chain formed by a sequence 3 and a sequence 4 in the sequence table as a target object, wherein the double-stranded nucleic acid specificity detection probe at normal temperature is composed of a single-stranded DNA shown in a sequence 8 in the sequence table and a single-stranded DNA shown in a sequence 9 in the sequence table (II type probe).
The desired double-stranded nucleic acid-specific detection probes W2 and W3 at room temperature were synthesized as shown in Table 2.
S2, adding a sample to be detected, exonuclease (lambda exo) and single-stranded binding protein (SSB) into a probe system containing the double-stranded nucleic acid specificity detection probe in the S1 at normal temperature to form a detection system, and recording fluorescence intensity;
s2-1 establishment of Probe System
Set up probe system (20 μ L): the reaction buffer solution environment is 1 x lambda exob, a double-stranded nucleic acid specific detection probe at normal temperature is added, the double-stranded nucleic acid specific detection probe at normal temperature is any one of W2-W3, and the concentration of each single-stranded DNA in the double-stranded nucleic acid specific detection probe at normal temperature in a system is 1 mu M. Make the total volume of the system equal to 20. Mu.L, the rest volume is ddH 2 And (4) supplementing by using oxygen.
An annealing procedure was applied to ensure that the strands of the oligonucleotides that make up the probes were recognized for proper binding. The annealing program was set as follows: at 90 ℃ for 2min; 1min at 85 ℃; at 80 ℃ for 1min;75 ℃ for 1min; 1min at 70 ℃; at 65 ℃ for 1min; at 60 ℃ for 1min; at 55 deg.C for 1min;50 ℃ for 1min; at 45 ℃ for 1min; at 40 ℃ for 1min; at 37 ℃ for 2min; at 16 ℃ for 1min; and keeping at 4 ℃.
A probe system 2 is established by using a double-stranded nucleic acid specificity detection probe W2 at normal temperature, and a probe system 3 is established by using a double-stranded nucleic acid specificity detection probe W3 at normal temperature.
S2-2 detection
20 μ L assay: mu.L of the above probe system (Probe System 2/Probe System 3) at a concentration of 1. Mu.M was taken to give a concentration of 100nM in the system, 10. Mu.L of the sample to be tested was added, lambda exo was added to give a concentration of 50U/mL in the system, and SSB was added to give a concentration of 500nM in the system. If the total volume of the system is less than 20 mu L, ddH is used 2 And (4) supplementing by using oxygen. And (3) putting the sample into a real-time fluorescence detector for detection at 37 ℃, setting the detection time interval to be 10 seconds per cycle, setting the cycle number to be 1600 times, and setting the detection time to be about 30 minutes to obtain the fluorescence intensity-time track. When the target nucleic acid sequence does not exist in the detection target, the fluorescence value has no obvious ascending change; if the fluorescence value increases significantly, the target nucleic acid sequence is considered to be present in the detection target.
S3, analyzing the fluorescence intensity recorded in the detection to determine whether a double-stranded DNA target exists in the sample to be detected:
the fluorescence-time trace when a target (double-stranded structure composed of T1-1 and T1-2) having a length equal to that of the probe-recognition region is detected is shown in graph A of FIG. 4, and the fluorescence-time trace when a target (double-stranded structure composed of T2-1 and T2-2) having a length longer than that of the probe-recognition region is detected is shown in graph B of FIG. 4. The results show that: if a double-stranded DNA target object exists in the sample to be detected, the fluorescence value is obviously increased no matter the target object is the target object with the same length as the probe identification area or the target object with the length longer than the probe identification area through detection of an I-type probe or a II-type probe; and if the target does not exist in the sample to be detected, the fluorescence value does not have obvious rising change.
The result shows that the composite probes can effectively distinguish double-stranded targets.
Example 3 detection method Using two types of composite probes for detection of artificially synthesized double-stranded target
The detection method of the two types of composite probes is used, the double-stranded DNA target object is a double strand consisting of T1-1 and T1-2 (the target object with the same length as the probe recognition area) or a double strand consisting of T2-1 and T2-2 (the target object with the length larger than the probe recognition area), the sample to be detected is the artificially synthesized double strand, and a blank control is set.
The embodiment is based on two types of composite probe detection, and the specific steps are as follows:
s1, designing a double-stranded nucleic acid specific detection probe at normal temperature according to a double-stranded DNA target object shown in Table 3, wherein the double-stranded nucleic acid specific detection probe at normal temperature used in the embodiment is a two-class composite probe, and the specific sequence is shown in Table 3:
TABLE 3 nucleic acid sequences
Note: in table 3, P represents phosphorylation and x represents thio.
FIG. 5 is a schematic diagram of the structure of a second type of composite probe, which is shown in Table 3 and is composed of three DNA single strands consisting of C1-C3:
c1 fluorescent probe (fluorophore-labeled single-stranded DNA, i.e., the fluorescent signal probe of FIG. 5, which can be 20-25 nucleotides in length);
a C2 quenching probe (quencher-labeled single-stranded DNA, i.e., the quenching signal probe of FIG. 5, which may be 20-25 nucleotides in length);
a C3 recognition probe (marked as C3-1), namely single-stranded DNA (deoxyribonucleic acid) which is formed by sequentially connecting a target probe binding domain, a fluorescent probe complementary domain and a quenching probe complementary domain according to the sequence from 5 'end to 3' end;
the C1 is stably combined with the complementary domain of the fluorescent probe in the C3; the C2 stably binds to a complementary domain of the quenching probe in the C3;
the probe satisfies that the fluorescence signal of the fluorescent group of C1 can be quenched by the quenching group of C2.
And the fluorescent group in the C1 is modified on the T base close to the 5' end, and is selected as FAM.
The quenching group in the C2 is modified at the 3 'end, the quenching group is BHQ1, the 5' end of the C2 is subjected to thio-modification, and the specific length of the thio-modification is 4-5 nucleotides;
the 5' end of the C3 is modified with a phosphate group.
The target probe binding domain is 45 nucleotides in length.
Wherein the molar ratio of C1, C2 and C3 is 1.
When a double strand consisting of T1-1 and T1-2 is selected as a target object, or a double strand consisting of T2-1 and T2-2 is selected as a target object, a set of double-stranded nucleic acid specific detection probe W4 at normal temperature is designed and obtained, wherein C3 is C3-1 in Table 3:
w4, a double strand consisting of a sequence 1 and a sequence 2 in a sequence table is taken as a target object, or a double strand consisting of a sequence 3 and a sequence 4 in the sequence table is taken as a target object, and the double-stranded nucleic acid specific detection probe at the normal temperature is composed of a single-stranded DNA shown in a sequence 10 in the sequence table, a single-stranded DNA shown in a sequence 11 in the sequence table, and a single-stranded DNA shown in a sequence 12 in the sequence table.
The desired probes for detecting double-stranded nucleic acid specificity at room temperature were synthesized according to Table 3.
S2, adding a sample to be detected, exonuclease (lambda exo) and single-stranded binding protein (SSB) into a probe system containing the double-stranded nucleic acid specificity detection probe in the S1 at normal temperature to form a detection system, and recording fluorescence intensity;
s2-1 establishment of Probe System
Establishing a Probe System(20. Mu.L): adding a double-stranded nucleic acid specific detection probe at normal temperature, wherein the double-stranded nucleic acid specific detection probe at normal temperature is W4, and the concentration of each single-stranded DNA in the double-stranded nucleic acid specific detection probe at normal temperature in a system is 1 mu M. Make the total volume of the system equal to 20. Mu.L, the rest volume is ddH 2 And (4) supplementing by using oxygen.
An annealing procedure was applied to ensure that the strands of the oligonucleotides making up the recognition probe were correctly bound. The annealing program was set as follows: at 90 ℃ for 2min; at 85 ℃ for 1min; at 80 ℃ for 1min; at 75 ℃ for 1min; 1min at 70 ℃; at 65 ℃ for 1min; at 60 ℃ for 1min; at 55 deg.C for 1min;50 ℃ for 1min; at 45 ℃ for 1min; 1min at 40 ℃; at 37 ℃ for 2min; at 16 ℃ for 1min; keeping at 4 ℃.
A probe system 4 is established by using a double-stranded nucleic acid specific detection probe W4 at normal temperature.
S2-2 detection
20 μ L assay: mu.L of the above probe system (probe system 4) having a concentration of 1. Mu.M was taken to have a concentration of 100nM in the system, 10. Mu.L of the sample to be tested was added, lambda exo was added to have a concentration of 50U/mL in the system, and SSB was added to have a concentration of 500nM in the system. If the total volume of the system is less than 20 mu L, ddH is used 2 And (4) supplementing by using oxygen. And (3) putting the sample into a real-time fluorescence detector for detection at the temperature of 37 ℃, setting the detection time interval to be 10 seconds and one cycle, setting the cycle number to be 1600 times, and setting the detection time to be about 30 minutes to obtain the fluorescence intensity-time track. When the target nucleic acid sequence does not exist in the detection target, the fluorescence value has no obvious ascending change; if the fluorescence value increases significantly, the target nucleic acid sequence is considered to be present in the detection target.
S3, analyzing the fluorescence intensity of the detection record to determine whether a double-stranded DNA target exists in the sample to be detected:
the fluorescence-time trajectory and gel electrophoresis characterization of the two types of composite probes when detecting a target object (double-stranded structure composed of T1-1 and T1-2) with the same length as the probe recognition region are shown in FIG. 6. The fluorescence-time traces and the gel electrophoresis characterization of the two types of composite probes for detecting a target object (a double strand consisting of T2-1 and T2-2) which is longer than the probe recognition area are shown in FIG. 7, and the probes are double-stranded nucleic acid specific detection probes W4. The result shows that if a double-stranded DNA target exists in the sample to be detected, the fluorescence value is obviously increased no matter the target is as long as the probe identification area or longer than the probe identification area; if the target does not exist in the sample to be detected, the fluorescence value does not have obvious rising change.
The results show that the two types of composite probes can effectively distinguish double-stranded targets.
Example 4 detection method Using two types of composite probes for detecting SARS-CoV-2-related Gene
The detection method of the two types of composite probes is used for detecting the N gene of SARS-CoV-2 or the E gene of SARS-CoV-2.
The experiment was done in the virus detection laboratory at the intermediate-day-friendly hospital. The samples to be tested used were as follows: a nasopharyngeal swab (from Kangji Biotechnology Co., ltd.) of 120 healthy people and COVID-19 patients (positive in nucleic acid detection) is taken for virus inactivation, a nucleic acid extraction kit (Tianlong technology, qEX-DNA/RNA virus) is used for extracting virus RNA, and reverse transcription and amplification are carried out on the RNA through an RT-RPA kit (twist Amp basic RPA kit). Obtaining double-chain nucleic acid fragments of N gene and E gene in SARS-CoV-2 virus respectively; and synthesizing a plasmid containing the virus N gene and the virus E gene, namely a sample to be detected, and setting a blank plasmid control.
The embodiment is based on two types of composite probe detection, and the specific steps are as follows:
s1, designing a double-stranded nucleic acid specific detection probe at normal temperature according to a double-stranded DNA target object shown in Table 4, wherein the double-stranded nucleic acid specific detection probe at normal temperature used in the embodiment is a two-class composite probe, and the specific sequence is shown in Table 4:
TABLE 4 nucleic acid sequences
Note: in table 4, P represents phosphorylation and x represents thio.
FIG. 5 is a schematic diagram of the structure of two types of complex probes, which are specifically shown in Table 4 and consist of three DNA single strands C1-C3:
a C1 fluorescent probe (fluorophore-labeled single-stranded DNA, i.e., the fluorescent signaling probe of FIG. 5, which may be 20-25 nucleotides in length);
a C2 quenching probe (quencher-labeled single-stranded DNA, i.e., the quenching signal probe of FIG. 5, which may be 20-25 nucleotides in length);
a C3 recognition probe, namely single-stranded DNA (deoxyribonucleic acid) which is formed by sequentially connecting a target probe binding domain, a fluorescent probe complementary domain and a quenching probe complementary domain according to the sequence from 5 'end to 3' end;
the C1 is stably combined with the complementary domain of the fluorescent probe in the C3; the C2 stably binds to a complementary domain of the quenching probe in the C3;
the probe satisfies that the fluorescence signal of the fluorescent group of C1 can be quenched by the quenching group of C2.
And the fluorescent group in the C1 is modified on the T base close to the 5' end, and is selected as FAM.
The quenching group in the C2 is modified at the 3 'end, the quenching group is BHQ1, the 5' end of the C2 is subjected to thio-modification, and the specific length of the thio-modification is 4-5 nucleotides;
the 5' end of the C3 is modified with a phosphate group.
The target probe binding domain is 45 nucleotides in length.
Wherein the molar ratio of C1, C2 and C3 is 1.
C3 when the N gene of SARS-CoV-2 is selected as a target is designated as C3-2; c3 when the E gene of SARS-CoV-2 was selected as a target was designated as C3-3. The method comprises the following specific steps:
the target gene is N gene, double chain (SARS-CoV-2 reverse transcription amplification product) composed of N1 and N2 is selected as target substance, a set of double chain nucleic acid specificity detection probe W5 at normal temperature is designed, and C3 is C3-2 in Table 4:
w5, a double strand consisting of a sequence 13 and a sequence 14 in a sequence table is taken as a target object, and the double-stranded nucleic acid specific detection probe at the normal temperature is composed of a single-stranded DNA shown in a sequence 10 in the sequence table, a single-stranded DNA shown in a sequence 11 in the sequence table, and a single-stranded DNA shown in a sequence 15 in the sequence table.
The target gene is E gene, double-chain (SARS-CoV-2 reverse transcription amplification product) composed of E1 and E2 is selected as target, a set of double-chain nucleic acid specificity detection probe W6 at normal temperature is designed, and the used C3 is C3-3 in Table 4:
w6, a double strand consisting of a sequence 16 and a sequence 17 in a sequence table is taken as a target, and the double-stranded nucleic acid specific detection probe at normal temperature is composed of a single-stranded DNA shown in a sequence 10 in the sequence table, a single-stranded DNA shown in a sequence 11 in the sequence table, and a single-stranded DNA shown in a sequence 18 in the sequence table.
The desired double-stranded nucleic acid specific detection probes at room temperature were synthesized according to Table 4.
S2, adding a sample to be detected, exonuclease (lambda exo) and single-stranded binding protein (SSB) into a probe system containing the double-stranded nucleic acid specificity detection probe in the S1 at normal temperature to form a detection system, and recording fluorescence intensity;
s2-1 establishment of Probe System
Set up probe system (20 μ L): the reaction buffer solution environment is 1 x lambda exo buffer, a double-stranded nucleic acid specific detection probe at normal temperature is added, the double-stranded nucleic acid specific detection probe at normal temperature is any one of W5-W6, and the concentration of each single-stranded DNA in the double-stranded nucleic acid specific detection probe at normal temperature in a system is 1 mu M. The total volume of the system was made equal to 20. Mu.L, the remaining volume was ddH 2 And (4) supplementing by using oxygen.
An annealing procedure was applied to ensure that the strands of the oligonucleotides that make up the probes were recognized for proper binding. The annealing program was programmed as follows: at 90 ℃ for 2min; 1min at 85 ℃; at 80 ℃ for 1min;75 ℃ for 1min; 1min at 70 ℃; at 65 ℃ for 1min; at 60 ℃ for 1min; at 55 deg.C for 1min; 1min at 50 ℃; at 45 ℃ for 1min; at 40 ℃ for 1min; at 37 ℃ for 2min; 1min at 16 ℃; keeping at 4 ℃.
A probe system 5 is established by using a double-stranded nucleic acid specific detection probe W5 at normal temperature, and a probe system 6 is established by using a double-stranded nucleic acid specific detection probe W6 at normal temperature.
S2-2 detection
20 μ L assay: mu.L of the above probe system (probe system 5/probe system 6) at a concentration of 1. Mu.M was taken to give a concentration of 100nM in the system, 10. Mu.L of the sample to be tested was added, lambda exo was added to give a concentration of 50U/mL in the system, and SSB was added to give a concentration of 500nM in the system. If the total volume of the system is less than 20 mu L, ddH is used 2 And (4) supplementing by using oxygen. And (3) putting the sample into a real-time fluorescence detector for detection at the temperature of 37 ℃, setting the detection time interval to be 10 seconds and one cycle, setting the cycle number to be 1600 times, and setting the detection time to be about 30 minutes to obtain the fluorescence intensity-time track. When the target nucleic acid sequence does not exist in the detection target, the fluorescence value has no obvious ascending change; if the fluorescence value increases significantly, the target nucleic acid sequence is considered to be present in the detection target.
S3, analyzing the fluorescence intensity of the detection record to determine whether a double-stranded DNA target exists in the sample to be detected:
the graph of fluorescence intensity-time trace in the presence of the N gene fragment in SARV-CoV-2 as the target is shown in B of FIG. 8, and the graph of fluorescence intensity-time trace in the presence of the E gene fragment in SARV-CoV-2 as the target is shown in A of FIG. 8. The results show that if a double-stranded DNA target exists in the sample to be detected, the fluorescence value is obviously increased; if the target does not exist in the sample to be detected, the fluorescence value does not have obvious rising change.
These results demonstrate that the probe of the present invention can directly detect a double-stranded nucleic acid target specifically at room temperature, can drive strand displacement reaction to form the property of the optimal substrate, recognizes and digests the 5' phosphate end of nucleic acid by using lambda exo, initiates digestion reaction of enzyme, and generates a fluorescent signal, and the signal strictly corresponds to the target nucleic acid sequence. The invention can directly identify the double-stranded nucleic acid target object at normal temperature (37 ℃), distinguish the target sequence from the non-target sequence with high reliability, greatly simplify the detection procedure of the double-stranded nucleic acid target object and reduce the difficulty of the on-site application of the double-stranded nucleic acid target object. The invention can combine polymerase-recombinase amplification (RPA), loop-mediated isothermal amplification (LAMP) and the like to realize high-sensitivity and high-specificity nucleic acid detection at normal temperature. The invention is also expected to be used for in-situ specific recognition and imaging of double-stranded nucleic acid in living cells.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
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Claims (9)
1. The probe for detecting the specificity of the double-stranded nucleic acid at the normal temperature is characterized by being any one of a single-stranded DNA type probe A, a first-class composite probe B and a second-class composite probe C:
the A single-stranded DNA type probe is a DNA single strand with a target probe binding domain, and the DNA single strand is marked with a fluorescent group and a quenching group; the fluorescent signal of the fluorescent group can be quenched by the quenching group;
the B type composite probe is I type or II type:
type I consists of two single-stranded DNAs B1-I and B2-I:
B1-I quenching probe;
the B2-I fluorescent group marked identification probe is formed by sequentially connecting a target binding domain and a quenching probe complementary domain; wherein the quenching probe complementary domain stably binds to said B1-I quenching probe;
type II consists of two single-stranded DNAs B1-II and B2-II:
B1-II fluorescent probes;
the B2-II quenching group marked identification probe is formed by sequentially connecting a target binding domain and a fluorescent probe complementary domain; wherein the fluorescent probe complementary domain is stably bound to the B1-II fluorescent probe;
the C second-class composite probe consists of three DNA single strands of C1-C3:
a C1 fluorescent probe;
a C2 quenching probe;
a C3 recognition probe, namely single-stranded DNA (deoxyribonucleic acid) which is formed by sequentially connecting a target probe binding domain, a fluorescent probe complementary domain and a quenching probe complementary domain according to the sequence from 5 'end to 3' end;
the C1 is stably combined with the complementary domain of the fluorescent probe in the C3; the C2 stably binds to a complementary domain of the quenching probe in the C3;
the double-stranded nucleic acid specificity detection probe at normal temperature comprises the steps of mixing the probe with a sample to be detected, lambda phage exonuclease and single-stranded binding protein, and detecting a fluorescent signal.
2. The probe for detecting the specificity of a double-stranded nucleic acid according to claim 1, wherein: the fluorescent group is FAM, and the quenching group is BHQ1.
3. The probe for detecting the specificity of a double-stranded nucleic acid according to claim 1, wherein: the 5' end of the A single-stranded DNA type probe is modified with a phosphate group; the quenching group in A is modified at the 3' end; the fluorescent group is modified on the T base;
the B-type composite probe: in the I type, the 5 'end of B2-I is modified with phosphate group, the fluorescent group of B2-I is modified on a T base close to the 3' end, the quenching group of B1-I quenching probe is modified on the 3 'end, and the 5' end of B1-I quenching probe is modified with sulfo; in the II type, the 5 'end of B2-II is modified with phosphate group, the quenching group of B2-II is modified at the 3' end, the fluorescent group of B1-II fluorescent probe is modified on the T basic group close to the 3 'end, and the 5' end of B1-II is modified with sulfo;
in the C-type and C-type composite probe, a fluorescent group in C1 is modified on a T base close to the 5' end; the quenching group in the C2 is modified at the 3' end, the 5' end of the C2 is modified with sulfo, and the 5' end of the C3 is modified with phosphate group.
4. The double-stranded nucleic acid specific detection probe at ordinary temperature according to any one of claims 1 to 3, wherein: the specific detection probe for the double-stranded nucleic acid at normal temperature is any one set of W1-W6:
w1, double chains consisting of SEQ ID No.1 and SEQ ID No.2 or double chains consisting of SEQ ID No.3 and SEQ ID No.4 are taken as target objects, and the double-stranded nucleic acid specificity detection probe at normal temperature is single-stranded DNA shown in SEQ ID No. 5;
w2, taking a double strand consisting of SEQ ID No.1 and SEQ ID No.2 as a target object, or taking a double strand consisting of SEQ ID No.3 and SEQ ID No.4 as a target object, wherein the double-stranded nucleic acid specificity detection probe at normal temperature consists of a single-stranded DNA shown by SEQ ID No.6 and a single-stranded DNA shown by SEQ ID No. 7;
w3, taking a double strand consisting of SEQ ID No.1 and SEQ ID No.2 as a target object, or taking a double strand consisting of SEQ ID No.3 and SEQ ID No.4 as a target object, wherein the double-stranded nucleic acid specificity detection probe at normal temperature consists of a single-stranded DNA shown in SEQ ID No.8 and a single-stranded DNA shown in SEQ ID No. 9;
w4, double chains consisting of SEQ ID No.1 and SEQ ID No.2 are taken as target objects, or double chains consisting of SEQ ID No.3 and SEQ ID No.4 are taken as target objects, and the double-stranded nucleic acid specificity detection probe at normal temperature is composed of single-stranded DNA shown by SEQ ID No.10, single-stranded DNA shown by SEQ ID No.11 and single-stranded DNA shown by SEQ ID No. 12;
w5, taking a double strand consisting of SEQ ID No.13 and SEQ ID No.14 as a target object, wherein the double-stranded nucleic acid specificity detection probe at normal temperature consists of a single-stranded DNA shown by SEQ ID No.10, a single-stranded DNA shown by SEQ ID No.11 and a single-stranded DNA shown by SEQ ID No. 15;
w6, taking a double strand consisting of SEQ ID No.16 and SEQ ID No.17 as a target, wherein the double-stranded nucleic acid specificity detection probe at normal temperature consists of a single-stranded DNA shown in SEQ ID No.10, a single-stranded DNA shown in SEQ ID No.11 and a single-stranded DNA shown in SEQ ID No. 18.
5. The method for detecting nucleic acid using the double-stranded nucleic acid specific detection probe at normal temperature according to any one of claims 1 to 4, comprising mixing the double-stranded nucleic acid specific detection probe at normal temperature with a sample to be detected, lambda phage exonuclease, and single-stranded binding protein, detecting a fluorescent signal, and judging whether the sample to be detected contains a target substance, wherein the judgment criteria are as follows: if a double-stranded DNA target exists in the sample to be detected, the fluorescence value is obviously increased; if the target does not exist in the sample to be detected, the fluorescence value does not have obvious rising change.
6. A product, characterized in that said product consists of X1 and X2; x1 is a double-stranded nucleic acid specific detection probe at normal temperature according to any one of claims 1 to 4, and X2 is a reagent and/or an instrument required for detection.
7. The product of claim 6, wherein the reagents required for detection comprise lambda phage exonuclease, single-stranded binding protein, and reaction buffer.
8. The product of claim 6, wherein the reagents required for detection are lambda phage exonuclease and single-stranded binding protein.
9. Use of a double-stranded nucleic acid specific detection probe according to any one of claims 1 to 4, or a method according to claim 5, or a product according to any one of claims 6 to 8 for the preparation of a nucleic acid detection reagent.
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