CN115725699A - Double-probe nucleic acid detection method - Google Patents
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Abstract
The invention relates to a method for detecting a target nucleic acid by using double probes. Specifically, the present invention provides a method for detecting a target sequence, comprising the steps of: (1) Hybridizing a capture strand to the target sequence to obtain a captured target sequence, wherein the capture strand is complementary to a first portion of the target sequence, (2) hybridizing the captured target sequence with the target strand, wherein the target strand recognizes a characteristic sequence of the target sequence, wherein the characteristic sequence is located in a region of the target sequence other than the first portion. The dual probe system of the invention can achieve a wide linear range and an extremely low detection limit.
Description
Technical Field
The invention relates to the field of nucleic acid detection, in particular to a double-probe nucleic acid detection method.
Background
Circulating tumor DNA (ctDNA) is a DNA fragment derived from tumor cells, free in peripheral blood. ctDNA can reflect tumor cell genome and change conditions thereof, and is considered to have very wide application prospects in the aspects of monitoring tumor gene mutation, monitoring tumor metastasis, helping treatment scheme selection, curative effect detection and the like. However, the ctDNA content in peripheral blood is extremely low, which causes a series of problems and difficulties such as high omission factor and difficult repetition of detection results. The development of an accurate and effective ctDNA detection method for accurately detecting the mutation from the tumor tissue in peripheral blood is an urgent need of current clinical tumor prevention and treatment, and has great application value.
The PIK3CA gene is a very important tumor gene and plays a key role particularly in Chinese breast cancer. Although the mutation of the PIK3CA gene is various, a mutation hotspot with higher frequency can be found in china, for example, the Histidine (H) mutation at the 1047 th position of the PIK3CA protein targeted by the present invention has higher frequency in the chinese breast cancer population. The site mutation can seriously affect the function of protein and has important relation with the generation and development of tumors. Correspondingly, the site also becomes a key target point of accurate diagnosis and treatment. Some effective therapeutic means have been found aiming at PIK3CA gene mutation, but the therapeutic schemes are based on the accurate detection of PIK3CA gene mutation.
In addition, the test of characteristic tumor sites in ctDNA is also an important index for tumorigenesis, development, detection and prognosis, and has a wide application range.
Disclosure of Invention
In a first aspect, the present invention provides a method of detecting a target sequence, comprising the steps of:
(1) Hybridizing a capture strand to the target sequence to obtain a captured target sequence, wherein the capture strand is complementary to a first portion of the target sequence,
(2) Hybridizing the captured target sequence with a target strand, said target strand recognizing a characteristic sequence of the target sequence, said characteristic sequence being located in a region of the target sequence other than the first portion,
optionally (3) detecting the presence or amount of the target strand.
In one or more embodiments, step (2) comprises: hybridizing the captured target sequence with a target strand, the target strand being complementary to a second portion of the target sequence, and the second portion comprising a signature sequence of the target sequence, the signature sequence being located in a region of the target sequence other than the first portion.
In one or more embodiments, the second portion is located in a region of the target sequence other than the first portion.
In one or more embodiments, the first portion does not include the signature sequence.
In one or more embodiments, the first portion and the second portion are adjacent.
In one or more embodiments, the signature sequence is a change in one or more bases relative to a reference.
In one or more embodiments, the signature sequence is a plurality of consecutive base changes from a reference.
In one or more embodiments, the signature sequence is a point mutation.
In one or more embodiments, the target sequence is ctDNA.
In one or more embodiments, the capture chain length is at least 5bp, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15bp.
In one or more embodiments, the capture strand is immobilized on a support.
In one or more embodiments, the capture strand has a linker. Preferably at the 3' end of the capture strand.
In one or more embodiments, the capture strand has a tag that facilitates separation or detection. Preferably at the 3' end of the capture strand.
In one or more embodiments, the tag is attached to the capture strand through the linker.
In one or more embodiments, the tag is-SH and the capture strand is attached to the support via the tag.
In one or more embodiments, the target strand length is at least 3bp, e.g., at least 4, 5, 6, 7, 8, 9bp.
In one or more embodiments, the recognition sequence of the signature sequence is located in the 3' region of the target strand. Preferably, the recognition sequence of the signature sequence is located in the 1 st to 5 th bases at the 3' end of the target strand.
In one or more embodiments, the recognition sequence for the signature sequence is located in the 5' region of the target strand. Preferably, the recognition sequence of the signature sequence is located in the 3 rd to 8 th bases of the 5' end of the target strand.
In one or more embodiments, the target strand is immobilized on a carrier.
In one or more embodiments, the target strand has an adaptor. Preferably at the 5' end of the target strand. For example, the linker is AAAAA.
In one or more embodiments, the target strands have tags that facilitate separation or facilitate detection. Preferably at the 5' end of the target strand.
In one or more embodiments, the tag is attached to the target strand through the adaptor.
In one or more embodiments, the tag is-SH and the target strand is attached to the conductive support via the tag.
In one or more embodiments, the capture strand comprises the sequence shown as SEQ ID NO. 2.
In one or more embodiments, the target strand comprises the sequence shown as SEQ ID No. 3.
In one or more embodiments, the capture strand of step (1) hybridizes to the target sequence at a temperature of 20-80 ℃, preferably 45-70 ℃, more preferably 50-70 ℃.
In one or more embodiments, the target strand of step (2) hybridizes to the target sequence at a temperature of 10-40 ℃, preferably 15-30 ℃, more preferably 22-28 ℃.
The invention also provides a capture strand capable of hybridising to a target sequence, said target sequence having a characteristic sequence, and the region of hybridisation of the capture strand to the target sequence does not comprise said characteristic sequence. Preferably, the capture strand comprises the sequence shown in SEQ ID NO 2.
The invention also provides a target strand capable of hybridizing with a target sequence, the target sequence has a characteristic sequence, and a hybridization region of the target strand and the target sequence comprises the characteristic sequence. Preferably, the target strand comprises the sequence shown as SEQ ID NO. 3.
In one or more embodiments, the capture strand, the target strand, or both are characterized as described in any embodiment of the first aspect of the invention.
The invention also provides a composition comprising a nucleic acid molecule comprising a capture strand and a target strand that are capable of hybridising to a target sequence, the target sequence having a signature sequence, wherein the capture strand is complementary to a first portion of the target sequence, the target strand is complementary to a second portion of the target sequence, and the second portion comprises the signature sequence of the target sequence, the signature sequence being located in a region of the target sequence other than the first portion.
In one or more embodiments, the second portion is located in a region of the target sequence other than the first portion.
In one or more embodiments, the first portion does not include the signature sequence.
In one or more embodiments, the signature sequence is a change in one or more bases relative to a reference.
In one or more embodiments, the signature sequence is a plurality of consecutive base changes from a reference.
In one or more embodiments, the signature sequence is a point mutation.
In one or more embodiments, the capture chain length is at least 5bp, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15bp.
In one or more embodiments, the capture strand has a linker. Preferably at the 3' end of the capture strand.
In one or more embodiments, the capture strand has a tag that facilitates separation or facilitates detection. Preferably at the 3' end of the capture strand.
In one or more embodiments, the tag is attached to the capture strand via the linker.
In one or more embodiments, the tag is-SH and the capture strand is attached to the support via the tag.
In one or more embodiments, the target strand length is at least 3bp, e.g., at least 4, 5, 6, 7, 8, 9bp, preferably 4-20bp, more preferably 6-15bp.
In one or more embodiments, the recognition sequence for the signature sequence is located in the 3' region of the target strand. Preferably, the recognition sequence of the signature sequence is located in the 1 st to 5 th bases at the 3' end of the target strand.
In one or more embodiments, the recognition sequence for the signature sequence is located in the 5' region of the target strand. Preferably, the recognition sequence of the signature sequence is located in the 3 rd to 8 th bases of the 5' end of the target strand.
In one or more embodiments, the target strand has an adaptor. Preferably at the 5' end of the target strand. For example, the linker is AAAAA.
In one or more embodiments, the target strand has a tag that facilitates separation or facilitates detection. Preferably at the 5' end of the target strand.
In one or more embodiments, the tag is attached to the target strand through the adaptor.
In one or more embodiments, the tag is-SH and the target strand is linked to the electrical conductor through the tag.
In one or more embodiments, the capture strand comprises the sequence shown as SEQ ID NO. 2.
In one or more embodiments, the target strand comprises the sequence shown as SEQ ID NO. 3.
The invention also provides a method for identifying the DNA characteristic sequence, which comprises the following steps:
(1) Hybridizing the capture strand with the target sequence to be detected to obtain a captured target sequence, wherein the capture strand is complementary with the target sequence,
(2) Contacting the captured target sequence with one or more target strands under conditions that allow nucleic acid hybridization, said target strands comprising sequences that recognize and hybridize to a DNA signature sequence,
(3) The DNA signature sequence of the target sequence is identified by the target strand that is capable of hybridizing to the target sequence.
In one or more embodiments, the recognition regions of the capture strand and the target strand on the target sequence do not overlap, preferably are adjacent.
In one or more embodiments, the signature sequence is a change in one or more bases relative to a reference.
In one or more embodiments, the signature sequence is a plurality of consecutive base changes from a reference.
In one or more embodiments, the signature sequence is a point mutation.
In one or more embodiments, the target sequence is ctDNA.
In one or more embodiments, the capture strand length is at least 5bp, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15bp.
In one or more embodiments, the capture strand has a linker. Preferably at the 3' end of the capture strand.
In one or more embodiments, the capture strand has a tag that facilitates separation or facilitates detection. Preferably at the 3' end of the capture strand.
In one or more embodiments, the tag is attached to the capture strand through the linker.
In one or more embodiments, the tag is-SH and the capture strand is attached to the support via the tag.
In one or more embodiments, the target strand length is at least 3bp, e.g., at least 4, 5, 6, 7, 8, 9bp, preferably 4-20bp, more preferably 6-15bp.
In one or more embodiments, the sequence on the target strand that recognizes the DNA signature sequence is a sequence complementary to the DNA signature sequence.
In one or more embodiments, the recognition sequence of the signature sequence is located in the 3' region of the target strand. Preferably, the recognition sequence of the signature sequence is located in the 1 st to 5 th bases at the 3' end of the target strand.
In one or more embodiments, the recognition sequence for the signature sequence is located in the 5' region of the target strand. Preferably, the recognition sequence of the signature sequence is located in the 3 rd to 8 th bases of the 5' end of the target strand.
In one or more embodiments, the target strand has an adaptor. Preferably at the 5' end of the target strand. For example, the linker is AAAAA.
In one or more embodiments, the target strand has a tag that facilitates separation or facilitates detection. Preferably at the 5' end of the target strand.
In one or more embodiments, the tag is linked to the target strand through the linker.
In one or more embodiments, the tag is-SH and the target strand is linked to the electrical conductor through the tag.
In one or more embodiments, the capture strand comprises the sequence shown as SEQ ID NO. 2.
In one or more embodiments, the target strand comprises the sequence shown as SEQ ID No. 3.
In one or more embodiments, the capture strand of step (1) hybridizes to the target sequence at a temperature of 20-80 ℃, preferably 45-70 ℃, more preferably 50-70 ℃.
In one or more embodiments, the target strand of step (2) hybridizes to the target sequence at a temperature of 10-40 ℃, preferably 15-30 ℃, more preferably 22-28 ℃.
An electrochemical biosensor for detecting a target sequence or identifying a DNA signature sequence, comprising a target strand as described in any embodiment herein attached to an electrical conductor.
In one or more embodiments, the electrical conductor is a carbon nanotube, preferably a multi-walled carbon nanotube.
In one or more embodiments, the carbon nanotubes are gold-platinum alloy loaded carbon nanotubes.
In one or more embodiments, the carbon nanotubes are PDA-modified carbon nanotubes.
In one or more embodiments, the electrical conductor is a multi-walled carbon nanotube-PDA-gold platinum alloy composite.
The invention also provides a system for detecting a target sequence or identifying a characteristic sequence of DNA, comprising: compositions, carbon nanotubes, buffers, electrodes as described herein.
In one or more embodiments, the carbon nanotubes are gold-platinum alloy loaded carbon nanotubes.
In one or more embodiments, the carbon nanotubes are PDA-modified carbon nanotubes.
In one or more embodiments, the carbon nanotubes are multi-walled carbon nanotube-PDA-gold platinum alloy composites.
In one or more embodiments, the buffer comprises H 2 O 2 。
In one or more embodiments, the electrodes are screen printed electrodes, preferably screen printed gold electrodes.
The invention also provides a device for detecting a target sequence or identifying a characteristic sequence of DNA, a system for detecting a target sequence or identifying a characteristic sequence of DNA and an amperometric detection device as described herein. Current sensing devices commonly used in electrochemistry are known in the art.
Drawings
FIG. 1 shows the optimization of hybridization conditions. (A) With different concentrations of H 2 O 2 CV of the multi-walled carbon nanotube PDA Au-Pt/SPCE of (a), (B) potential, (C) capture strand incubation temperature and (D) target strand incubation temperature (n = 5).
Figure 2 shows the accuracy of the two-stage probe combination looking at (a) the current response and (B) the corresponding current response histogram of the probe combination for different DNA sequences (n = 5). Blank: blank control, CM-DNA: perfect mismatch DNA, SM-DNA: single base mismatch DNA, T-DNA: target-DNA.
Fig. 3 shows (a) amperometric responses and (B) calibration curves of the biosensor for different concentrations of ctDNA (n = 5).
Detailed Description
The inventors have found that the specificity and sensitivity of detection can be significantly improved by using both the capture strand and the target strand (also referred to herein in some embodiments as the signal strand) when detecting a nucleic acid signature sequence (e.g., a mutation). An electrochemical biosensor designed based on this principle can detect target sequence sequences with a wide linear range and an extremely low detection limit.
As used herein, "target nucleic acid" and "target sequence" are used interchangeably and refer to a test nucleic acid that contains or may contain a characteristic sequence.
Nucleic acid detection as used herein is primarily meant to include DNA detection. The DNA may be DNA of any origin, e.g. genomic, cfDNA, ctDNA. The detection method of the invention has high specificity and sensitivity, and can realize the detection of trace DNA in the sample.
As used herein, "signature sequence" refers to any particular sequence of interest, such as a modified base (e.g., a methylated base), a mutated base, a mutated sequence, a SNP, a sequence having a particular structure (e.g., a loop, a hairpin). The mutation may be a point mutation or a continuous mutation, and preferably a point mutation.
As used herein, "hybridization", "complementary" is based on the relatively stable interaction between two nucleic acids that are base paired. Hybridization mainly refers to hybridization under stringent conditions. "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding a denaturing agent upon hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1%; or (3) hybridization only when the identity between two sequences is at least 90% or more, preferably 95% or more. Typically, complementation includes complete complementation and partial complementation. Moreover, hybridization does not imply a perfectly complementary pair.
As used herein, "recognition" includes hybridization and also includes identification of a particular base or sequence. Such as the identification and characterization of methylated cytosines. The present invention encompasses the identification of a particular base or sequence as is conventional in the art.
The term "capture" refers to the association of a molecule of interest (e.g., a DNA fragment) with a capture molecule (e.g., a capture strand) by some chemical or physical method. The term "target strand" refers to a nucleic acid molecule that recognizes a characteristic sequence of a target sequence. Target strands with a detectable signal (e.g., linked to a detectable species) are referred to as signal strands. The probe molecules herein comprise a capture strand and a target strand.
To facilitate subsequent manipulations, such as enrichment or detection, the nucleic acid molecules herein (e.g., probe molecules including capture and target strands) have elements that can facilitate their relative separation from the original system, along with the molecules with which they are associated, and/or elements that can facilitate their detection.
For example, to facilitate enrichment, the probe molecule can be attached to a solid support via a covalent bond. Alternatively, a linker and/or a specific group can be added to both ends of the probe molecule, and these linker or specific group can be covalently linked to the solid support. By enriching the solid phase support, the capture strand and the target sequence hybridized thereto can be enriched. The skilled person is aware of conventional solid supports and of suitable linkers or specific groups.
As another example, to facilitate detection, the probe molecule can be linked to the detectable moiety by a covalent bond. Alternatively, a linker and/or a specific group may be added to both ends of the probe molecule, which linker or specific group is capable of covalently linking to the detectable species. The probes and their hybridization sequences can be characterized or quantified by a detectable signal or change thereof provided by the detectable species. For example, the detectable species may be a detectable label, such as a fluorophore, that releases a signal that is recognized by another detection system, or may be a carrier, such as an electrical conductor, that causes a change in a signal, such as an electrical signal. The person skilled in the art is aware of the customary detectable substances and also of the suitable linkers or specific groups.
The method of the invention is based on a two-probe hybridization process. In a first aspect, the present invention provides a method of detecting a target sequence, comprising the steps of: (1) Hybridizing a capture strand to a target sequence to obtain a captured target sequence, wherein the capture strand is complementary to a first portion of the target sequence, (2) hybridizing a target strand to the captured target sequence, wherein the target strand recognizes a characteristic sequence of the target sequence, wherein the characteristic sequence is located in a region of the target sequence other than the first portion. Whereby the presence or amount of the target strand is indicative of the presence or amount of the target sequence. In certain embodiments, the target strand is complementary to a second portion of the target sequence, and the second portion comprises a signature sequence of the target sequence, the signature sequence being located in a region of the target sequence other than the first portion. I.e., the hybridizing region of the capture strand does not include the sequence characteristic of the target sequence. The method of detecting a target sequence can be used to detect the presence of the target sequence in a sample when the sequence characteristic of the target sequence is known. For example, it is examined whether a certain SNP site or a mutation site or region of DNA has a certain base or bases.
In exemplary embodiments, the second portion is located in a region of the target sequence other than the first portion (e.g., the first portion and the second portion are contiguous), and do not overlap. However, the present invention also encompasses a scheme in which the first portion overlaps the second portion, as long as the hybridization region of the capture strand does not include the characteristic sequence of the target sequence.
The length of the capture strand is not limited as long as it can stably hybridize and enrich for the target sequence. In some embodiments, the capture strand is at least 5bp, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15bp, preferably 10-30bp, more preferably 15-25bp.
The capture strand and target sequence hybridized thereto can be enriched using any method known in the art for enriching nucleic acids. For example, as described above, the capture strand may have a linker (e.g., AAAAA) that facilitates enrichment or detection, preferably at the 3' end of the capture strand. Through this linker, the capture strand is linked to a tag (e.g., -SH) that facilitates enrichment or facilitates detection. The capture strand is attached to a support (e.g., a gold substrate surface) via the tag.
The length of the target strand is also not limited as long as it can stably hybridize to the signature sequence and be detected. In some embodiments, the target strand is at least 3bp, e.g., at least 4, 5, 6, 7, 8, 9bp, preferably 4-20bp, more preferably 6-15bp. The target strand recognizes the signature sequence by one or more of its bases.
The inventors have found that the recognition sequence of the signature sequence (e.g., its complement) can be located anywhere on the target strand, such as in the 5 'region, the middle, or the 3' region, where 5', 3', and the middle refer to about one-third of the 5 'end, about one-third of the 3' end, and about one-third of the middle of the sequence. Preferably, the recognition sequence of the signature sequence is located in the 3 'region of the target strand, for example in the 1 st to 5 th bases, preferably 2 nd or 3 rd bases, at the 3' end of the target strand; alternatively, the recognition sequence of the signature sequence is located in the 5 'region of the target strand, for example in the 3 rd to 8 th bases, preferably the 6 th or 7 th bases, at the 5' end of the target strand.
The presence or amount of the target strand can be detected using any method known in the art for qualitatively or quantitatively detecting nucleic acids. For example, as described above, the target strand may have a linker (e.g., AAAAA) that facilitates enrichment or detection, preferably located at the 5' end of the target strand. Through this linker, the target strand is linked to a tag (e.g., -SH) that facilitates enrichment or detection. The target strand is linked to a detectable species (e.g., an electrical conductor) via the tag.
In exemplary embodiments, the detection is performed using the methods of the present invention in combination with an electrochemical method. The obtained multi-walled carbon nanotube-PDA-gold platinum alloy (multi-walled carbon nanotube PDA Au-Pt) can respond to H by uniformly dispersing gold platinum alloy nanoparticles on the multi-walled carbon nanotube-PDA 2 O 2 Reduction of (2). The capture strand is immobilized on the surface of the screen-printed gold electrode and captures the target DNA, and when the target DNA is hybridized with the target strand, the carbon conductor on the target strand provides electric signal detection.
The test for detecting the target sequence or identifying the characteristic sequence of the multi-wall carbon nanotube PDA Au-Pt is carried out at the potential of-10V to 5V, preferably-5V to 3V, -2V to 1V, -0.5V to 0.5V, -0.2V to 0.2V and-0.2V to 0V. In an exemplary embodiment, the multi-walled carbon nanotube PDA Au-Pt detection target sequence or identification of its characteristic sequence is performed at a potential of-0.1V.
Herein, the temperature at which the capture strand hybridizes to the target sequence is 20 to 80 ℃, preferably 45 to 70 ℃, more preferably 50 to 70 ℃. In an exemplary embodiment, the temperature at which the capture strand hybridizes to the target sequence is 64 ℃.
Herein, the temperature at which the target strand hybridizes to the target sequence is 10-40 ℃, preferably 15-30 ℃, more preferably 22-28 ℃. In an exemplary embodiment, the temperature at which the target strand hybridizes to the target sequence is 25 ℃.
The invention also provides a method for identifying the DNA characteristic sequence, which comprises the following steps: obtaining a captured target sequence by hybridizing a capture strand to a target sequence to be detected, wherein the capture strand is complementary to the target sequence, (2) contacting the captured target sequence with one or more target strands under conditions that allow nucleic acid hybridization, said target strands comprising a sequence that recognizes and hybridizes to a DNA signature sequence, and (3) identifying the DNA signature sequence of the target sequence by the target strand that hybridizes to the target sequence. The hybridized region of the capture strand does not include sequences characteristic of the target sequence that may be present. Methods for identifying a characteristic sequence of DNA can be used to determine whether DNA in a sample has a certain characteristic sequence at a site or region. The method can also screen a population for which signature sequences (e.g., which mutations or SNPs) are present at a site or region based on high throughput assays.
The recognition regions of the capture strand and the target strand on the target sequence, if present, do not overlap (e.g., are adjacent). However, the present invention also encompasses a scheme in which the recognition regions of both overlap, as long as the hybridization region of the capture strand does not include the characteristic sequence of the target sequence. In the method of identifying a characteristic sequence of DNA, the other characteristics (length, linker, hybridization temperature, etc.) of the capture strand and the target strand are as described in the method of detecting a target sequence described herein before.
The method of identifying the DNA signature sequence may also be combined with electrochemical methods for testing, the appropriate potentials and other test parameters being similar to those described above for the method of detecting the target sequence.
The present invention also provides the above-mentioned capture strand and target strand as nucleic acid molecules, wherein the capture strand and target strand are capable of hybridizing to a target sequence having a characteristic sequence, and the region of hybridization of the capture strand to the target sequence does not comprise said characteristic sequence, while the region of hybridization of the target strand to the target sequence comprises said characteristic sequence.
The invention also provides a composition comprising a capture strand and a target strand as described above which are capable of hybridising to a target sequence, wherein the capture strand is complementary to a first portion of the target sequence, the target strand is complementary to a second portion of the target sequence, and the second portion comprises a characteristic sequence of the target sequence, which characteristic sequence is located in a region of the target sequence other than the first portion.
In an exemplary embodiment, the target sequence is a DNA fragment comprising a coding region for amino acid 1047 of the PIK3CA gene. Since the amino acid can be histidine or arginine, the reading frame of the nucleic acid can be CAT or CGT (i.e. a characteristic sequence), and the sequence is shown as the 251 st nucleic acid of SEQ ID NO. 1 (rs 121913279). The method can efficiently and accurately detect whether the PIK3CA gene in the sample is CAT or CGT at the position.
In this exemplary embodiment, the methods of detection herein separate the probe sequence into a capture strand and a target strand. The first segment is called Capture Chain (CP), the sequence is shown in SEQ ID NO:2, the length is 19 bases, the first segment is complementary with the 230 th-248 th base sequence in SEQ ID NO:1 in reverse direction, namely complementary with a segment of sequence in PIK3CA gene. The function of the method is to capture the corresponding sequence in the ctDNA fragment of the PIK3CA gene in the target. The capture chain can be immobilized on a substrate surface by attaching various groups at the 3' end, such as 5 consecutive groups of A plus thiol (-SH).
The second segment is called a target strand (e.g., signal strands, SP)), and the sequence is shown in SEQ ID NO:3, 9 bases in length, and is characterized in that the 7 th base is designed to be a C that pairs with mutant PIK3CA rather than a T that pairs with wild type, and such design allows the Signal strand to hybridize exactly with mutant ctDNA without hybridizing with the wild type PIK3CA gene sequence. The signal chain may provide signal detection or output by attachment of various groups at the 5' end, such as 5 consecutive A plus thiol (-SH) groups attached to a carbon rod to provide electrical signal detection.
The inventors have adopted an electrochemical method comprising using a screen-printed electrode and adding a multi-walled carbon nanotube-PDA-gold platinum alloy to obtain a detectable electrical signal by immobilization of the capture strand on the surface of the screen-printed gold electrode, recognition of the capture strand to the target DNA, and hybridization of the target DNA to the signal strand. The probe composition of the present invention can obtain 1X 10 -15 mol.L-1 to 1X 10 - 8 The wide linear range of mol.L-1, the detection limit is as low as 3.16 multiplied by 10 -16 mol. L-1. Methods for electrochemical detection using screen-printed electrodes and carbon nanotubes are well known in the art.
Examples
In this example, the inventors used an electrochemical method to uniformly disperse the gold-platinum alloy nanoparticles on the multi-walled carbon nanotube-PDA, and obtained the multi-walled carbon nanotube-PDA-gold-platinum alloy pair H 2 O 2 The reduction of (2) has good response, and the current response is greatly enhanced. Including a capture chainThe double-probe sandwich structure is successfully formed through step-by-step reaction and comprises the steps of fixing a capture probe on the surface of a screen-printed gold electrode, recognizing a capture strand and target DNA (T-DNA) and hybridizing the T-DNA and the target strand.
Optimization of the Experimental conditions
In order to obtain better ctDNA detection performance, the concentration is 1 × 10 -6 At the T-DNA concentration of mol/L, the experimental conditions including the applied potential and the hybridization temperature are optimized.
Prior to selecting the optimal applied potential that may have a significant impact on the electrochemical signal of the biosensor, CV tests were performed at pH =7.4PBS to approximately determine the approximate location of the optimal potential. FIG. 1, A shows that the multi-walled carbon nanotube PDA-Au-Pt modified electrode pair has different concentrations of H 2 O 2 The CV response characteristic of (1). The reduction peak in the range of-0V to 0.2V and the broad oxidation peak in the range of 0.4V to 0.7V are attributed to the electrical signal of Pt 48. Furthermore, with H 2 O 2 The concentration is increased, the reduction peak current is obviously enhanced, and the MWCNTs-PDA-Au-Pt is proved to be in H pair 2 O 2 Has good response. The final optimum potential was further evaluated by amperometry (I-t), as shown in FIG. 1, B, multi-walled carbon nanotubes PDA Au-Pt at-0.1V vs. H 2 O 2 Exhibiting a maximum response. Thus, the subsequent experiments were carried out at a potential of-0.1V.
In the two hybridization processes of the capture chain and the T-DNA, and the T-DNA and the target chain for recognizing, the DNA culture temperature influencing the DNA hybridization reaction is optimized. As can be seen from FIG. 1, C, the current signal reaches a maximum at 64 ℃ and thus in the subsequent experiments T-DNA was incubated with CPs at 64 ℃. In addition, fig. 1 d shows that the current signal increases sharply in the temperature range of 15 ℃ to 25 ℃, but rapidly decreases when the temperature exceeds 25 ℃. According to the results, 25 ℃ was chosen as the hybridization temperature of the T-DNA and the target strand in all experiments.
Detection of two-stage probe combination
In actual detection, mutation positions are arranged at a plurality of base positions at the 5' end, the middle part or the 3' end of the signal chain, and through specific experiments and comparison, the mutation position is finally preferred to be at the 7 th base from the 5' end of the signal chain. Adjusting the concentration of the probe to test the sensitivity of the system; single-mismatch and full-mismatch interfering sequences that differ from the target DNA by only one base were designed to test the accuracy of the system.
5 consecutive A and thiol groups were added to the capture and target strands (SEQ ID NO:2 and SEQ ID NO: 3) as follows:
the concentration of the test sample was normalized to 1X 10 -8 mol/L. As a result, as shown in FIG. 2, the two-stage probe combination can accurately detect the mutated target DNA, and the signal amounts of the single-base mismatched DNA and the perfect mismatched DNA are similar to those of the blank control, and almost no signal is generated.
Detection Range and detection Limit
Under the optimal experimental conditions (potential-0.1V, incubation temperature of T-DNA and capture chain is 64 ℃, and incubation temperature of T-DNA and target chain is 25 ℃), the sandwich type electrochemical biosensor using the multi-wall carbon nano-tube PDA-Au-Pt as the marker is used for detecting target sequences (T-DNA) with different concentrations, and the capture chain, the target chain and the target sequence are shown as above.
The results are shown in FIG. 3. In fig. 3, a, when 5mM H is added to PBS buffer of pH =7.4 2 O 2 The current response increases proportionally with increasing T-DNA concentration at-0.1V potential. As shown in FIG. 3, B, the biosensor shows a range from 1X 10 -15 mol L -1 To 1 x 10 -8 mol L -1 Wide linear range of (2), detection limit is as low as 3.16X 10 -16 mol L -1 (S/N = 3). The regression equation for the calibration curve may be expressed as Δ I (μ =0.299logC +4.80 (R) 2 = 0.996), wherein C is the concentration of T-DNA.
Compared with other T-DNA electrochemical analysis methods reported in the past literature, the biosensor based on the double-probe sandwich scheme of the invention obtains a wider linear range and a lower detection limit.
SEQUENCE LISTING
<110> technical research institute of seafood industry
<120> double-probe nucleic acid detection method
<130> 213598
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 501
<212> DNA
<213> Homo sapiens
<400> 1
taacatcatt tgctccaaac tgaccaaact gttcttatta cttataggtt tcaggagatg 60
tgttacaagg cttatctagc tattcgacag catgccaatc tcttcataaa tcttttctca 120
atgatgcttg gctctggaat gccagaacta caatcttttg atgacattgc atacattcga 180
aagaccctag ccttagataa aactgagcaa gaggctttgg agtatttcat gaaacaaatg 240
aatgatgcac rtcatggtgg ctggacaaca aaaatggatt ggatcttcca cacaattaaa 300
cagcatgcat tgaactgaaa agataactga gaaaatgaaa gctcactctg gattccacac 360
tgcactgtta ataactctca gcaggcaaag accgattgca taggaattgc acaatccatg 420
aacagcatta gaatttacag caagaacaga aataaaatac tatataattt aaataatgta 480
aacgcaaaca gggtttgata g 501
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Capture chain
<400> 2
gcatcattca tttgtttca 19
<210> 3
<211> 9
<212> DNA
<213> Artificial Sequence
<220>
<223> target Strand
<400> 3
ccatgacgt 9
Claims (10)
1. A method of detecting a target sequence comprising the steps of:
(1) Hybridizing a capture strand to the target sequence to obtain a captured target sequence, wherein the capture strand is complementary to a first portion of the target sequence,
(2) Hybridizing the captured target sequence with a target strand that recognizes a signature sequence of the target sequence, the signature sequence being located in a region of the target sequence other than the first portion.
2. The method of claim 1, wherein the target strand is complementary to a second portion of the target sequence, and the second portion comprises a signature sequence of the target sequence, the signature sequence being located in a region of the target sequence other than the first portion,
preferably, the first and second electrodes are formed of a metal,
the signature sequence is a change in one or more bases relative to a reference, and/or
The target sequence is ctDNA.
3. The method of claim 1 or 2, wherein the method further has one or more characteristics selected from the group consisting of:
the length of the capture strand is at least 5bp,
the capture strand has a tag that facilitates separation or detection,
the length of the target chain is at least 3bp,
the target strand recognizes the signature sequence by one or more bases of its 3 'region, preferably, the target strand recognizes the signature sequence by one or more of bases 1 to 5 of its 3' end,
the target strand recognizes the signature sequence by one or more bases of its 5 'region, preferably, the target strand recognizes the signature sequence by one or more of bases 3-8 of its 5' end,
the target strand has a linker attached thereto,
the target strands have tags for separation or detection,
the temperature at which the capture strand of step (1) hybridizes to the target sequence is 20-80 ℃, preferably 45-70 ℃, more preferably 50-70 ℃, and
the temperature at which the target strand of step (2) hybridizes to the target sequence is 10-40 ℃, preferably 15-30 ℃, more preferably 22-28 ℃.
4. A method for identifying a DNA signature sequence comprising the steps of:
(1) Hybridizing the capture strand with the target sequence to be detected to obtain a captured target sequence, wherein the capture strand is complementary with the target sequence,
(2) Contacting the captured target sequence with one or more target strands under conditions that allow nucleic acid hybridization, said target strands comprising a sequence that recognizes and hybridizes to a DNA signature sequence,
(3) Identifying the DNA signature sequence of the target sequence by the target strand capable of hybridizing to the target sequence,
preferably, the first and second electrodes are formed of a metal,
the signature sequence is a change in one or more bases relative to a reference, and/or
The target sequence is ctDNA.
5. The method of claim 4,
the capture chain length is at least 5bp, and/or
The capture strand has a tag for separation or detection.
6. The method of claim 4,
the target strand is at least 3bp in length, and/or
The target strand recognizes the signature sequence by one or more bases of its 3 'region, preferably, the target strand recognizes the signature sequence by one or more of bases 1-5 of its 3' end, and/or
The target strand recognizes the signature sequence by one or more bases of its 5 'region, preferably, the target strand recognizes the signature sequence by one or more bases of 3-8 bases of its 5' end, and/or
The target strand has a tag for separation or detection, and/or
The temperature at which the capture strand of step (1) hybridizes to the target sequence is 20-80 ℃, preferably 45-70 ℃, more preferably 50-70 ℃, and/or
The temperature at which the target strand of step (2) hybridizes to the target sequence is 10-40 deg.C, preferably 15-30 deg.C, and more preferably 22-28 deg.C.
7. A composition comprising a nucleic acid molecule comprising a capture strand and a target strand that are hybridizable to a target sequence, the target sequence having a signature sequence, wherein the capture strand is complementary to a first portion of the target sequence, the target strand is complementary to a second portion of the target sequence, and the second portion comprises the signature sequence of the target sequence, the signature sequence being located in a region of the target sequence other than the first portion,
preferably, the first and second electrodes are formed of a metal,
the second portion is located in a region of the target sequence other than the first portion, and/or
The signature sequence is a change in one or more bases relative to a reference.
8. The composition of claim 7, wherein the composition further has one or more characteristics selected from the group consisting of:
the length of the capture strand is at least 5bp,
the capture strand has a tag for separation or detection,
the target strand is at least 3bp in length,
the recognition sequence of the characteristic sequence is located in the 3 'region of the target strand, preferably in the 1 st to 5 th bases at the 3' end of the target strand,
the complement of the signature sequence is located in the 5 'region of the target strand, preferably in the 3 rd to 8 th bases of the 5' end of the target strand,
the target strand has a tag for separation or detection.
9. A system for detecting a target sequence or identifying a characteristic sequence of DNA, comprising: the composition, carbon nanotube, buffer, electrode of claim 8,
preferably, the first and second liquid crystal display panels are,
the carbon nanotubes are loaded with a gold-platinum alloy, and/or
The carbon nanotubes are PDA-modified carbon nanotubes, preferably multi-walled carbon nanotube-PDA-gold platinum alloy composite, and/or
The buffer comprises H 2 O 2 And/or
The electrodes are screen printed electrodes, preferably screen printed gold electrodes.
10. A system or device for detecting a target sequence or identifying a DNA signature comprising the system for detecting a target sequence or identifying a DNA signature of claim 9 and an amperometric detection device.
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