CN116287107A - Nucleic acid probe and application method thereof - Google Patents

Nucleic acid probe and application method thereof Download PDF

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CN116287107A
CN116287107A CN202111574315.3A CN202111574315A CN116287107A CN 116287107 A CN116287107 A CN 116287107A CN 202111574315 A CN202111574315 A CN 202111574315A CN 116287107 A CN116287107 A CN 116287107A
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nucleic acid
acid probe
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黄菁
徐璨
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Guangdong Fapon Biotech Co Ltd
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Guangdong Fapon Biotech Co Ltd
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Priority to PCT/CN2022/136654 priority patent/WO2023116408A1/en
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Abstract

A nucleic acid probe is disclosed that is suitable for binding to more than two target sequences that differ in sequence and that has more than one base mismatch with at least one target sequence. The nucleic acid probe can improve the overall amplification efficiency, reduce the probability of generating dimer, secondary structure and nonspecific amplification products, and reduce the synthesis cost of the probe.

Description

Nucleic acid probe and application method thereof
Technical Field
The invention relates to a biological detection method, in particular to a nucleic acid probe.
Background
The nucleic acid probe is a nucleic acid sequence (DNA or RNA) with a detection label and known sequence and complementary to the target gene. The nucleic acid probe is combined with the target gene through molecular hybridization to generate hybridization signals, so that the target gene can be displayed from a vast genome. According to the hybridization principle, the nucleic acid sequence as a probe must have at least the following two conditions: (1) if the label is double-stranded, the label must be denatured and (2) the label should be easily detected. It may include the entire gene or may be only a portion of the gene; either DNA itself or RNA transcribed from it.
Among them, the TaqMan probe is a use form of a nucleic acid probe. It is a sequence specific, fluorescently labeled oligonucleotide with a Reporter fluorophore (R) at the 5 'end, typically FAM, VIC, HEX, TET, ROX, CY, CY3, etc., and a quenching fluorophore (Q) at the 3' end, typically TAMRA, BHQ1, BHQ2, etc. The principle of qPCR by TaqMan probe method is that a pair of primers are added during PCR amplification, and a specific TaqMan fluorescent probe is additionally added, wherein the specific TaqMan fluorescent probe is specifically combined with a template, and the combining site is between the two primers. When the probe is complete, the space distance between the reporter fluorescent group and the quenching fluorescent group is very short, and the fluorescent signal emitted by the reporter fluorescent group can be absorbed by the quenching fluorescent group, so that the instrument can not detect the fluorescent signal. In the PCR extension stage, taq DNA polymerase synthesizes new chain along the direction from 5' to 3' of template chain, when Taq DNA polymerase reaches the probe binding site, 5' -3' exonuclease activity of Taq DNA polymerase cuts off the reporter fluorescent group connected with 5' end of the probe, so that the reporter fluorescent group and the quenching fluorescent group are separated, fluorescence is emitted, the number of the cut fluorescent molecules is proportional to the number of PCR products, and therefore, the purpose of detecting the amplification amount of the PCR products can be achieved by detecting the fluorescence intensity in a PCR reaction system.
Although TaqMan probe method has a plurality of advantages, the synthesis cost of the probe is more expensive and the experimental cost is higher.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a nucleic acid probe and a use method thereof. The nucleic acid probe can reduce the cost of probe synthesis.
The invention is realized in the following way:
embodiments of the present invention provide a nucleic acid probe that is suitable for binding to two or more target sequences that differ in sequence and that has one or more base mismatches with at least one of the target sequences.
The embodiment of the invention also provides application of the nucleic acid probe in detection of target sequences, wherein the application is used for being matched with more than two pairs of primers.
The embodiment of the invention also provides a nucleic acid composition which comprises the nucleic acid probe and more than two pairs of primers.
The embodiment of the invention also provides application of the accounting probe or the nucleic acid composition in preparation of a kit for HPV detection and/or typing.
The embodiment of the invention also provides a reagent or a kit for detecting a target sequence, which comprises the nucleic acid probe or the nucleic acid composition.
The embodiment of the invention also provides a method for detecting the target sequence, which comprises the step of adding the nucleic acid composition into a reaction system for detecting the target sequence.
The beneficial effects of the embodiment of the invention are as follows:
the invention provides a nucleic acid composition, which comprises a nucleic acid probe and more than two pairs of primers, wherein the nucleic acid probe and the more than two pairs of primers are matched to detect more than two target sequences, and have more than one base mismatch with at least one target sequence, the 5 'end of the nucleic acid probe is marked with a report fluorescent group, and the 3' end of the nucleic acid probe is marked with a quenching fluorescent group; the nucleic acid composition can improve the overall amplification efficiency, reduce the probability of generating dimers, secondary structures and nonspecific amplification products, and reduce the synthesis cost of probes.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of pairing of a nucleic acid probe with three templates presented on the probe design software.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Embodiments of the present invention provide a nucleic acid probe that can bind to two or more target sequences that differ in sequence and that has one or more base mismatches with at least one of the target sequences. Referring to the principle of fig. 1, a nucleic acid probe may bind to three target sequences having different sequences, and the three target sequences have a certain similarity in sequence, so that the nucleic acid probe may bind to all of the three target sequences through a mismatch of several bases. The target sequence may be a sequence segment of different virus subtypes, which may be used as a typing basis, and the virus may be detected and/or typed by binding of a nucleic acid probe to the target sequence.
The term "probe" refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that contains a particular nucleotide sequence by design or selection that allows it to hybridize, under defined predetermined stringency, to a "target nucleic acid", in this case a target sequence in the L1 gene. The "probe" may be referred to as a "detection probe", meaning that it detects a target nucleic acid.
And designing mismatch sites by utilizing a base complementary pairing principle, wherein the mismatch sites simultaneously meet the detection of a plurality of target sequences. The artificial addition, subtraction or substitution of bases in the binding region allows a probe sequence to bind to several subtypes of the target band through different mismatched patterns. Reduces the probability of generating dimer, secondary structure and nonspecific amplification product, and reduces the synthesis cost of the probe.
In some embodiments, the base mismatch of the nucleic acid probe and the target sequence comprises any of the following: a substitution of one or several nucleotides, a deletion of one or several nucleotides or an insertion of one or several nucleotides. In particular, it refers to the phenomenon that nucleic acid probes have non-complementary base pairing with respect to the sequence of the target sequence, i.e., the bases on the probe are not complementary to the corresponding bases on the target sequence.
According to a typical embodiment of the invention, the nucleic acid probe has a mismatch of at most 7 bases; in some embodiments, there is at most a 6 base mismatch; in some embodiments, there is at most a 5 base mismatch. Specific forms of mismatches include the presence of substitutions, deletions, and insertions of the nucleic acid probe relative to the sequence of the target sequence.
In some embodiments, the nucleic acid probe is labeled with a reporter fluorophore at the 5 'end and a quencher fluorophore at the 3' end. Specifically, the reporter fluorophore is FAM, VIC, HEX, TET, ROX, CY or CY3, and the quencher fluorophore is TAMRA, BHQ1 or BHQ2.
In some embodiments, the nucleic acid probe is also used in combination with two or more pairs of primers for amplifying two or more different target sequences as described above. The primer pair is used for amplifying target sequences, and the nucleic acid probe can be combined with various amplified target sequences.
The term "primer" is used herein as known to those skilled in the art and refers to an oligomeric compound, primarily an oligonucleotide, but also to a modified oligonucleotide, which is capable of "priming" DNA synthesis by a template dependent DNA polymerase, i.e. e.g. the 3 '-end of the oligonucleotide provides a free 3' -OH group, where more "nucleotides" can be bound thereto by the template dependent DNA polymerase, creating 3 'to 5' phosphodiester bonds, thereby using deoxynucleoside triphosphates, and thereby releasing pyrophosphates.
In alternative embodiments, the length of the nucleic acid probe may be arbitrarily selected in the range of 20nt to 40nt; preferably, the length is 23nt to 35nt, such as 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, 30nt, 31nt, 32nt, 33nt, 34nt, 35nt.
In alternative embodiments, the Tm value of the nucleic acid probe can be arbitrarily selected within the range of 60℃to 75 ℃; preferably, the Tm value thereof is 65-70℃such as 65℃66℃67℃68℃69℃70 ℃.
In alternative embodiments, the reporter fluorophore may be selected from FAM, VIC, HEX, TET, ROX, CY or CY3 and the quencher fluorophore may be selected from TAMRA, BHQ1 or BHQ2.
The application of the nucleic acid probe can be used for HPV detection or HPV typing detection;
alternatively, HPV typing is selected from 33, 52, 58, 39, 45, 68, 56, 66, 31, 35, 51 or 59;
alternatively, HPV typing is selected from 33, 52, 58; alternatively, HPV typing is selected from 39, 45, 68; alternatively, HPV typing is selected from 56, 66; alternatively, HPV typing is selected from 31, 35.
The present invention provides a nucleic acid composition comprising at least one nucleic acid probe as described in any of the preceding examples or embodiments, and said two or more pairs of primers.
The embodiment of the invention provides application of the nucleic acid probe and the nucleic acid composition in preparation of a kit for HPV detection and/or typing;
alternatively, HPV typing is selected from 33, 52, 58, 39, 45, 68, 56, 66, 31, 35, 51 or 59;
alternatively, HPV typing is selected from 33, 52, 58; alternatively, HPV typing is selected from 39, 45, 68; alternatively, HPV typing is selected from 56, 66; alternatively, HPV typing is selected from 31, 35.
The present invention provides a reagent or kit for detecting a target sequence, comprising a nucleic acid probe or a nucleic acid composition as described in any of the previous examples or embodiments.
The embodiment of the invention provides a method for detecting a target sequence, which comprises the following steps: adding the nucleic acid composition of any of the foregoing examples or embodiments to a reaction system for detecting a target sequence;
in a preferred embodiment, the reaction system for detecting the mutation of the target gene is a fluorescent real-time quantitative PCR reaction system.
Examples
Example 1
By analyzing the gene region and market research, the L1 region gene sequence of the high-risk HPV is selected as a target sequence, and primers are designed in the selected sequence region by using Primer Premier 5.0 software, so that the detection method is established and optimized. By evaluating the accuracy, sensitivity and specificity, a fluorescence PCR method is established for detecting or typing the high-risk HPV genes.
Primer pair information designed:
Figure BDA0003424773150000051
Figure BDA0003424773150000061
description: 56/66-R,31/35-R,39/68-R,33/58-R are each independently represented as follows, e.g., 56/66-R represents a sequence of either 56-R or 66-R, and the remainder are not described in detail.
Example 2
Designing and obtaining nucleic acid probes:
nucleic acid probe design was performed using Bioeidit software, and some bases were added or substituted in the region near the middle of the nucleic acid probe binding L1 amplification segment, allowing one probe sequence to bind several subtypes of the target band through different mismatch forms (see the principle shown in fig. 1), resulting in the following probe sequences, with appropriate fluorophores FAM, and quencher BHQ1 at the time of synthesis:
Figure BDA0003424773150000062
example 3
Single amplification detection: for different types of nucleic acid probes and a corresponding pair of primers, detection is performed in a single tube, each experimental group contains positive samples of different types, and each experimental group is provided with two parallel experiments:
1.1 detection sample nucleic acid extraction: the sample was 10000 CP/. Mu.L of an artificially synthesized positive plasmid sample containing 10 ng/. Mu.L of human genome, and after extraction using TIANGEN magnetic bead virus RNA/DNA extraction kit, 5. Mu.L was taken for on-machine detection.
1.2 preparation of 25 μLPCR System with the above primer probe combinations:
Figure BDA0003424773150000071
two multiplex well parallel experiments were set up for each nucleic acid probe and primer set.
1.3 on-machine amplification procedure:
95 ℃ for 15min; (ROX and HEX fluorescence values are collected at 95 ℃ for 10s and 52 ℃ for 40 s; and 45 cycles are collected at 72 ℃ for 20 s); 25s at 25 DEG C
1.4 CT values were obtained from the amplification curves, and specific CT value data were analyzed together with the following examples.
Example 4
Multiplex amplification detection: all types of nucleic acid probes and primers were mixed in the same tube for detection, but 12 experimental groups were set, each containing a different type of positive sample, and each experimental group was set up for two parallel experiments.
Nucleic acid extraction, system configuration and setup of the computer program were performed with reference to example 3. The results obtained were combined with the single-dose test of example 3.
Figure BDA0003424773150000081
From the results, the final amplification results of the single amplification and the multiplex amplification are consistent, and the amplification effects are not greatly different for the 12 HPV subtypes. The primer probe combination can be used and has good effect. The primer probes do not interfere with each other. More importantly, the probe can be combined with a plurality of target sequences simultaneously to realize HPV typing. Since typing detection can be achieved under a 12-weight system, at least 1 probe and two or three corresponding pairs of target sequence primers can be adopted to detect corresponding HPV typing.
SEQUENCE LISTING
<110> Guangdong Fit biological Co., ltd
<120> a nucleic acid probe and method of using the same
<130> 2021
<160> 26
<170> PatentIn version 3.5
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Claims (10)

1. A nucleic acid probe adapted to bind to two or more target sequences of different sequences and having one or more base mismatches with at least one of the target sequences.
2. The nucleic acid probe of claim 1, wherein the base mismatch comprises any of the following: a substitution of one or several nucleotides, a deletion of one or several nucleotides or an insertion of one or several nucleotides;
alternatively, there is at most a 7 base mismatch; alternatively, there is at most a 6 base mismatch; alternatively, there is at most a 5 base mismatch.
3. The nucleic acid probe of claim 1, wherein the nucleic acid probe is used in combination with two or more pairs of primers for amplifying two or more target sequences having different sequences.
4. The nucleic acid probe according to claim 1, wherein the length thereof is 20nt to 40nt; preferably, the length thereof is 23nt to 35nt.
5. The nucleic acid probe according to claim 1, wherein the Tm value is 60 to 75 ℃; preferably, the Tm value thereof is 65-70 ℃.
6. The nucleic acid probe of claim 1, wherein the nucleic acid probe is labeled with a reporter fluorophore at the 5 'end and a quencher fluorophore at the 3' end;
alternatively, the reporter fluorophore is FAM, VIC, HEX, TET, ROX, CY5 or CY3 and the quencher fluorophore is TAMRA, BHQ1 or BHQ2.
7. A nucleic acid composition comprising the nucleic acid probe of claim 1 to 6 and two or more pairs of primers of claim 3.
8. Use of the nucleic acid probe according to claims 1 to 6, the nucleic acid composition according to claim 7 for the preparation of a kit for HPV detection and/or typing;
alternatively, HPV typing is selected from 33, 52, 58, 39, 45, 68, 56, 66, 31, 35, 51 or 59;
alternatively, HPV typing is selected from 33, 52, 58; alternatively, HPV typing is selected from 39, 45, 68; alternatively, HPV typing is selected from 56, 66; alternatively, HPV typing is selected from 31, 35.
9. A reagent or kit for detecting a target sequence, comprising the nucleic acid probe according to any one of claims 1 to 6 or the nucleic acid composition according to claim 7.
10. A method of detecting a target sequence, comprising: adding the nucleic acid probe according to any one of claims 1 to 6 or the nucleic acid composition according to claim 7 to a reaction system for detecting a target sequence;
preferably, the reaction system for detecting the mutation of the target gene is a fluorescent real-time quantitative PCR reaction system.
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