WO2023116408A1 - Nucleic acid probe and method for using same - Google Patents

Abstract

The present invention provides a nucleic acid probe, which is suitable for binding two or more target sequences having different sequences and has one or more base mismatch with at least one target sequence. According to the nucleic acid probe, the overall amplification efficiency can be improved, the probability of generation of dimers, secondary structures and non-specific amplification products can be reduced, and the probe synthesis cost can be reduced.

Description

A nucleic acid probe and method of use thereof

Cross References to Related Applications

This disclosure claims the priority of the Chinese patent application with the application number "202111574315.3" and the title "A Nucleic Acid Probe and Its Using Method" filed with the China Patent Office on December 21, 2021, the entire contents of which are incorporated herein by reference. In public.

technical field

The present disclosure relates to a biological detection method, in particular to a nucleic acid probe.

Background technique

A nucleic acid probe is a nucleic acid sequence (DNA or RNA) with a detection label and a known sequence that is complementary to the target gene. Nucleic acid probes combine with the target gene through molecular hybridization to generate a hybridization signal, which can display the target gene from the vast genome. According to the principle of hybridization, the nucleic acid sequence used as a probe must at least meet the following two conditions: ① it should be single-stranded, if it is double-stranded, it must be denatured first, and ② it should have a label that can be easily detected. It can include the entire gene, or just a part of it; it can be the DNA itself, or the RNA transcribed from it.

Among them, TaqMan probe is a used form of nucleic acid probe. It is a sequence-specific, fluorescently labeled oligonucleotide, and its 5' end is labeled with a reporter fluorescent group (Reporter, R), generally FAM, VIC, HEX, TET, ROX, CY5, CY3 and other fluorescent groups , the 3' end is labeled with a quencher fluorescent group (Quencher, Q), generally TAMRA, BHQ1, BHQ2, etc. The principle of TaqMan probe method qPCR is to add a specific TaqMan fluorescent probe while adding a pair of primers during PCR amplification. The probe specifically binds to the template, and its binding site is between the two primers. . When the probe is intact, the spatial distance between the reporter fluorescent group and the quencher fluorescent group is very close, and the fluorescent signal emitted by the reporter fluorescent group will be absorbed by the quencher fluorescent group, so that the instrument cannot detect the fluorescent signal. During the PCR elongation phase, Taq DNA polymerase synthesizes a new strand along the direction of the template strand from 5' to 3', and when Taq DNA polymerase reaches the probe binding site, the 5'-3' exonucleic acid Dicer activity cuts off the reporter fluorescent group attached to the 5' end of the probe, so that the reporter fluorescent group and the quencher fluorescent group are separated to emit fluorescence. The number of cut fluorescent molecules is proportional to the number of PCR products. Therefore, The purpose of detecting the amplification amount of PCR products can be achieved by detecting the fluorescence intensity in the PCR reaction system.

Although the TaqMan probe method has many advantages, the cost of probe synthesis is relatively high, and the experimental cost is relatively high.

Contents of the invention

The present disclosure provides a nucleic acid probe and a method of using the same.

The present disclosure provides a nucleic acid probe, which is suitable for binding two or more target sequences with different sequences, and has more than one base mismatch with at least one target sequence.

The present disclosure provides the application of the nucleic acid probe in cooperation with more than two pairs of primers to detect a target sequence.

The present disclosure provides a nucleic acid composition, which includes the nucleic acid probe and the two or more pairs of primers.

The present disclosure provides the application of the nucleic acid probe or nucleic acid composition in preparing a kit for HPV detection and/or typing.

The present disclosure provides a reagent or kit for detecting a target sequence, which includes the nucleic acid probe or the nucleic acid composition.

The present disclosure provides a method for detecting a target sequence, adding the nucleic acid composition into a reaction system for detecting the target sequence.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only Some implementations of the present disclosure are shown, so it should not be regarded as a limitation on the scope. For those skilled in the art, other relevant Attached picture.

Figure 1 is the principle of pairing nucleic acid probes with three templates presented on the probe design software.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. If the conditions are not specified in the embodiment, it shall be carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Definition of Terms

As used herein, the term "probe" refers to a synthetic or biologically produced nucleic acid (DNA or RNA) that is designed or selected to contain a specific nucleotide sequence that allows it to interact with a "target nucleic acid" under a defined, predetermined stringency. ”, that is, the target sequence hybridization in the L1 gene. A "probe" may be referred to as a "detection probe", meaning that it detects a target nucleic acid.

As used herein, the term "primer" is used as known to those skilled in the art, and refers to oligomeric compounds, mainly oligonucleotides, but also modified oligonucleotides, which are capable of template-dependent DNA polymerization Enzymatic "priming" of DNA synthesis, i.e. e.g. the 3'-end of an oligonucleotide presents a free 3'-OH group, where more "nucleotides" can be bound to it by a template-dependent DNA polymerase, establishing 3' to 5' phosphodiester linkage, thereby using deoxynucleoside triphosphates, and thereby releasing pyrophosphate.

The present disclosure provides a nucleic acid probe, which can bind to two or more target sequences with different sequences, and has more than one base mismatch with at least one target sequence. Referring to the principle in Figure 1, a nucleic acid probe can bind to three target sequences with different sequences, and the three target sequences have a certain similarity in sequence, so that the nucleic acid probe can be combined with three target sequences through a few base mismatches. target sequences are bound. It should be noted that the target sequence can be a sequence segment in different subtypes of the virus that can be used as a basis for typing, and the virus can be detected and/or typed through the binding of the nucleic acid probe to the target sequence.

In one or more exemplary embodiments, the nucleic acid probe utilizes the principle of complementary base pairing to design mismatch sites to simultaneously meet the detection of multiple target sequences. Artificially increase, decrease or replace some bases in the binding region, so that a probe sequence can combine with several subtypes of target bands through different mismatching forms. Reduce the probability of dimers, secondary structures, and non-specific amplification products, and reduce the cost of probe synthesis.

In one or more exemplary embodiments, the base mismatch between the nucleic acid probe and the target sequence includes any of the following: a substitution of one or several nucleotides, a deletion of one or several nucleotides, or a or insertions of a few nucleotides. Specifically refers to the phenomenon that the nucleic acid probe has non-complementary base pairing with respect to the sequence of the target sequence, that is, the base on the probe is not complementary to the corresponding base on the target sequence

According to one or more exemplary embodiments of the present disclosure, the nucleic acid probe has at most 7 base mismatches; in one or more exemplary embodiments, at most 6 base mismatches; In some embodiments, there is a mismatch of up to 5 bases. Mismatches include substitutions, deletions, and insertions of the nucleic acid probe relative to the sequence of the target sequence.

In one or more exemplary embodiments, the 5' end of the nucleic acid probe is labeled with a reporter fluorescent group, and the 3' end is labeled with a quencher fluorescent group. Optionally, the reporter fluorophore is FAM, VIC, HEX, TET, ROX, CY5 or CY3, and the quencher fluorophore is TAMRA, BHQ1 or BHQ2.

In one or more exemplary embodiments, the nucleic acid probe is also used in conjunction with two or more pairs of primers, and the primers are used to amplify the above two or more target sequences with different sequences. The primer pair is used to amplify the target sequence, and the nucleic acid probe can be combined with various amplified target sequences.

In one or more exemplary embodiments, the length of the nucleic acid probe can be arbitrarily selected within the range of 20nt to 40nt; the length is, for example, 23nt to 35nt, such as 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt , 30nt, 31nt, 32nt, 33nt, 34nt, 35nt.

In one or more exemplary embodiments, the Tm value of the nucleic acid probe can be arbitrarily selected within the range of 60-75°C; the Tm value is, for example, 65-70°C, such as 65°C, 66°C, 67°C, 68°C, 69°C, 70°C.

In one or more exemplary embodiments, the reporter fluorophore can be selected from FAM, VIC, HEX, TET, ROX, CY5 or CY3, and the quencher fluorophore can be selected from TAMRA, BHQ1 or BHQ2.

The present disclosure provides the application of nucleic acid probe or nucleic acid composition, which can be used for HPV detection or HPV typing detection;

In one or more exemplary embodiments, the HPV type is selected from 33, 52, 58, 39, 45, 68, 56, 66, 31, 35, 51 or 59;

In one or more exemplary embodiments, HPV typing is selected from 33, 52, 58; Optionally, HPV typing is selected from 39, 45, 68; Optionally, HPV typing is selected from 56, 66 ; Optionally, the HPV type is selected from 31,35.

The present disclosure provides a nucleic acid composition, which includes at least one nucleic acid probe described in any embodiment, and the above two pairs of primers.

The present disclosure provides nucleic acid probes and the application of the nucleic acid composition in the preparation of HPV detection and/or typing kits;

In one or more exemplary embodiments, the HPV type is selected from 33, 52, 58, 39, 45, 68, 56, 66, 31, 35, 51 or 59;

In one or more exemplary embodiments, HPV typing is selected from 33, 52, 58; In one or more exemplary embodiments, HPV typing is selected from 39, 45, 68; In one or more In various exemplary embodiments, the HPV type is selected from 56, 66; in one or more exemplary embodiments, the HPV type is selected from 31, 35.

The present disclosure provides a reagent or kit for detecting a target sequence, which includes the nucleic acid probe or the nucleic acid composition described in any of the embodiments or embodiments.

The present disclosure provides a method for detecting a target sequence, which includes: adding the nucleic acid composition described in any of the above embodiments to a reaction system for detecting a target sequence;

In one or more exemplary embodiments, the reaction system for detecting target gene mutation is a fluorescent real-time quantitative PCR reaction system.

The present disclosure provides a nucleic acid composition, the nucleic acid composition includes a nucleic acid probe and more than two pairs of primers, used in conjunction to detect more than two target sequences, and has more than one base mismatch with at least one target sequence , the 5' end of the nucleic acid probe is labeled with a reporter fluorophore, and the 3' end is labeled with a quencher fluorophore; the nucleic acid composition can improve the overall amplification efficiency and reduce dimers, secondary structures, and non-specific amplification The probability of product generation reduces the cost of probe synthesis.

Example

Design and acquisition of embodiment 1 nucleic acid primer

Through the analysis of the gene region and market research, the gene sequence of the L1 region of high-risk HPV was selected as the target sequence, and the Primer Premier 5.0 software was used to design primers in the selected sequence region, and the detection method was established and optimized. By evaluating its accuracy, sensitivity and specificity, a fluorescent PCR method was established for the detection or typing of high-risk HPV genes.

Designed primer pair information:

Explanation: 56/66-R, 31/35-R, 39/68-R, and 33/58-R independently represent the following meanings, for example, 56/66-R means that this sequence is both 56-R and 66-R , and the rest will not be repeated.

Example 2 The design and acquisition of nucleic acid probes Use Bioeidit software to design nucleic acid probes, add or replace some bases in the middle region of the nucleic acid probe combined with the L1 amplification segment, so that a probe sequence can pass through different The mismatched form is combined with several subtypes of target bands (refer to the principle shown in Figure 1) to obtain the following probe sequence, which is synthesized with a suitable fluorescent group FAM and a quencher group BHQ1:

Example 3 HPV single amplification detection

Single-plex amplification detection: For different types of nucleic acid probes and the corresponding pair of primers, they are detected in a single tube, each experimental group contains different types of positive samples, and each experimental group is set up with two tubes in parallel experiment:

1.1 Nucleic acid extraction from test samples: 10,000 CP/μL of artificially synthesized positive plasmid samples were used for the samples, which contained 10 ng/μL of human genome. After extraction using the TIANGEN Magnetic Bead Method Viral RNA/DNA Extraction Kit, 5 μL was taken for detection on the machine.

1.2 Prepare a 25 μL PCR system using the above primer and probe combinations:

For each nucleic acid probe and primer set, two replicate wells were set up for parallel experiments.

1.3 Computer amplification program:

95°C for 15min; (95°C for 10s; 52°C for 40s to collect ROX and HEX fluorescence values; 72°C for 20s) 45cycle; 25°C for 25s

1.4 The CT value is obtained through the amplification curve, and the CT value data will be summarized and analyzed together with the next embodiment.

Example 4 HPV Multiple Amplification Detection

Multiple amplification detection: All types of nucleic acid probes and primers are mixed in the same tube for detection, but 12 experimental groups are set up, each experimental group contains different types of positive samples, and each experimental group is set up with two tubes for parallel experiments.

Referring to Example 3, nucleic acid extraction, system configuration, and computer program setting were performed. The results obtained and the single detection in Example 3 were combined for analysis.

From the results, the final amplification results of single amplification and multiple amplification were consistent, and there was little difference in the amplification effects of the 12 HPV subtypes. It is proved that the primer-probe combination can be used and the effect is good. More importantly, a probe of the present disclosure can be combined with multiple target sequences simultaneously to realize HPV typing. Since the typing detection can be realized under the 12-plex system, the corresponding HPV typing can also be detected by using at least one probe and corresponding two or three pairs of target sequence primers.

While examples of the present disclosure have been illustrated and described, it should be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the disclosure. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this disclosure.

Industrial Applicability

The present disclosure provides a nucleic acid composition, kit and application thereof. The nucleic acid composition prolongs the shelf life of reagents for nucleic acid amplification, solves the problem of expensive synthesis of multiple nucleic acid primers and probes, can eliminate the synthesis of unnecessary probes, effectively reduces the cost of probe synthesis, and has broad application prospects and higher market value.

Claims (10)

1. A nucleic acid probe, which is suitable for binding two or more target sequences with different sequences, and has more than one base mismatch with at least one target sequence.
2. The nucleic acid probe according to claim 1, wherein the base mismatch includes any of the following: substitution of one or several nucleotides, deletion of one or several nucleotides, or one or several nucleosides Insertion of nucleotides;

   Preferably, there is a mismatch of at most 7 bases; preferably, there is a mismatch of at most 6 bases; preferably, there is a mismatch of at most 5 bases.
3. The nucleic acid probe according to claims 1-2, wherein the nucleic acid probe is used in combination with two or more pairs of primers, and the primers are used to amplify the above two or more target sequences with different sequences.
4. The nucleic acid probe according to claims 1-3, wherein the length of the nucleic acid probe is 20nt-40nt; more preferably 23nt-35nt.
5. The nucleic acid probe according to claims 1-4, wherein the Tm value of the nucleic acid probe is 60-75°C; more preferably 65-70°C.
6. The nucleic acid probe according to claims 1 to 5, wherein the 5' end of the nucleic acid probe is labeled with a reporter fluorescent group, and the 3' end is labeled with a quenched fluorescent group;

   Preferably, the reporter fluorescent group is FAM, VIC, HEX, TET, ROX, CY5 or CY3, and the quencher fluorescent group is TAMRA, BHQ1 or BHQ2.
7. A nucleic acid composition, comprising the nucleic acid probe according to claims 1-6, and two or more pairs of primers according to claim 3.
8. Application of the nucleic acid probe as claimed in claims 1 to 6 and the nucleic acid composition as claimed in claim 7 in the preparation of a kit for HPV detection and/or typing;

   Preferably, the HPV type is selected from 33, 52, 58, 39, 45, 68, 56, 66, 31, 35, 51 or 59;

   Preferably, HPV typing is selected from 33, 52, 58; Preferably, HPV typing is selected from 39, 45, 68; Preferably, HPV typing is selected from 56, 66; Preferably, 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 1-6 or the nucleic acid composition according to claim 7.
10. A method for 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 into a reaction system for detecting a target sequence;

    Preferably, the reaction system for detecting target gene mutation is a fluorescent real-time quantitative PCR reaction system.

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