CN106929593B - In-situ nucleic acid detection method - Google Patents
In-situ nucleic acid detection method Download PDFInfo
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Abstract
The invention discloses an in-situ nucleic acid detection method, which is based on the specific recognition of a target sequence by a double-connection probe and the amplification of a signal by rolling circle amplification and can be used for in-situ detection of one or more target nucleic acids. The novel method produces a specific detection signal with a high signal-to-noise ratio, enabling the detection of nucleic acid target sequences in a sample.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to an in-situ nucleic acid detection method.
Background
Nucleic acid in situ detection techniques enable localized detection of DNA and RNA in cell and tissue samples. The traditional in situ detection technology of nucleic acid is the in situ hybridization technology (ISH), the method is to hybridize the labeled detection probe and the target nucleic acid, the unbound probe is washed away, the detection probe specifically hybridized with the target nucleic acid is detected by the label, thereby realizing the detection of the target nucleic acid. Wherein the single-molecule fluorescence in situ hybridization technique (smFISH) enables in situ detection of a single RNA molecule by hybridizing multiple detection probes on the single RNA molecule to obtain a sufficiently strong signal. Compared with the traditional ISH, the single-molecule fluorescence in situ hybridization technology has higher sensitivity and specificity.
Amplification of the signal can also be achieved by rolling circle amplification, provided that the target nucleic acid molecule to be detected is converted into a circular nucleic acid molecule which specifically corresponds to the target nucleic acid molecule. The single-molecule RNA in-situ detection technology by the locking-type probe method is a method which can detect single-molecule RNA and can also detect base variation. The method specifically hybridizes two sections of regions of a padlock probe formed by a single-stranded DNA with a target sequence of target nucleic acid, when bases at two ends of the probe are completely complementary with the target sequence, two ends of the probe are connected by DNA ligase to form a circular DNA molecule, then the circular DNA molecule is amplified through Rolling Circle Amplification (RCA), and finally the detection of the target RNA is realized by detecting an amplification product by using a detection probe. The detection of base mutation is usually carried out by simultaneously competing and hybridizing two or more padlock probes with specific detection probe binding regions with a target sequence of target nucleic acid, finally binding the complete complementary padlock probes to the target sequence and further connecting and amplifying the complete complementary padlock probes, and obtaining the information of variant bases through the specific detection probes.
Disclosure of Invention
The invention aims to provide an in-situ nucleic acid detection method, which highly integrates an in-situ hybridization technology, a rolling circle amplification technology and a double-connection probe technology and realizes the detection of a nucleic acid target sequence in a sample.
The technical scheme of the invention is as follows:
an in situ nucleic acid detection method comprising the steps of:
(1) subjecting at least one pair of ligation probes to specific hybridization complementarity with a target sequence in a sample to be tested; each pair of ligation probes comprises an upstream probe and a downstream probe: the 3 'end of the upstream probe is provided with an upstream recognition sequence which is specifically complementary with the upstream sequence of the target sequence, and the 5' end is provided with an upstream loop-forming connecting template complementary sequence; the 5 'end of the downstream probe is provided with a downstream recognition sequence which is specifically complementary with the downstream sequence of the target sequence, and the 3' end is provided with a downstream loop-forming connecting template complementary sequence; the upstream sequence and the downstream sequence of the target sequence do not overlap with each other;
(2) connecting an upstream recognition sequence and a downstream recognition sequence which are specifically hybridized and complemented with a target sequence in a sample to be detected by ligase to form at least one first sequence;
(3) enabling at least one looping connecting template to be simultaneously and specifically hybridized and complemented with an upstream looping connecting template complementary sequence and a downstream looping connecting template complementary sequence on two sides of at least one first sequence, wherein the upstream looping connecting template complementary sequence and the downstream looping connecting template complementary sequence are not overlapped with each other relative to the looping connecting template;
(4) connecting the upstream cyclization connecting template complementary sequence which is specifically hybridized and complemented with the cyclization connecting template with the downstream cyclization connecting template complementary sequence by ligase to obtain at least one circular template;
(5) performing rolling circle amplification on the at least one annular template to obtain at least one rolling circle amplification product;
(6) and hybridizing the at least one rolling circle amplification product with at least one detection probe to detect.
In a preferred embodiment of the invention, the target sequence is a cDNA produced by reverse transcription of DNA, RNA or RNA.
In a preferred embodiment of the invention, the detection probe is modified with a fluorescent label, a chromogenic label, an enzymatic label, a radioactive label, a magnetic label or a luminescent density label.
In a preferred embodiment of the invention, at least one pair of ligation probes comprises natural and/or non-natural nucleotides.
In a preferred embodiment of the invention, the ligation templates contain natural and/or non-natural nucleotides.
In a preferred embodiment of the present invention, the test sample comprises cells in culture, tissues and tissue sections.
In a preferred embodiment of the present invention, the ligase includes T4 DNA ligase and Ampligase DNA ligase.
The invention has the beneficial effects that: the invention can effectively realize the detection of target nucleic acid, and has high signal-to-noise ratio and good specificity.
Drawings
FIG. 1 is a schematic view of a pair of ligation probes according to the present invention.
Fig. 2 is a schematic diagram of the principle of the present invention.
FIG. 3 is a graph showing the results of detection in example 1 of the present invention.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1 human skin fibroblasts are experimental samples to examine the double-ligation probe method for in situ detection of cellular ACTB mRNA.
Firstly, cell culture and fixation:
human skin fibroblasts were cultured in DMEM (containing 10% FBS) for 24-48h, then trypsinized to suspension cells, plated on sterile slides with polylysine on the surface, and re-cultured for 12-24 h. Washing with DEPC-PBS for 3 × 3 min; fixing with 3% PFA prepared by DEPC-PBS at room temperature for 30 min; after washing once more with DEPC-PBS, the reaction mixture was washed with gradient ethanol: 70%, 85%, 100% dehydration treatment, each for 5 min. And (5) air drying.
mRNA in situ reverse transcription of (di) ACTB
The cell membrane of human fibroblasts was subjected to membrane-penetrating perforation, and 0.1M HCl was added to the sample and incubated at room temperature for 5 min. In situ reverse transcription of ACTB mRNA was performed after three washes with DEPC-PBS-Tween 20. In the reaction system, using ACTB mRNA contained in the sample as a template, 50uL of a reverse transcription mixture containing 1mM dNTP, 1. mu.M reverse transcription primer (5'-CTGACCCATGCCCACCATCACGCCC-3', SEQ ID NO 01), 20 u/uLReverted H minus M-MuLV reverse transcriptase (Fermentas), 1 u/uL RiboLock RNase inhibitor (Fermentas) and RT buffer (Fermentas) at a final concentration of 1X was added, and mRNA molecules were reverse transcribed into cDNA in situ. After incubation for 3 hours at 37 ℃ the cells were washed three times with DEPC-PBS-Tween 20.
(III) in situ nucleic acid detection, as shown in FIG. 1 and FIG. 1, specifically comprising the steps of:
(1) double ligation Probe hybrid ligation
50uL of ligation mixture containing Ampligase buffer (Epicenter) at a final concentration of 1X, 0.1. mu.M upstream probe, 0.1. mu.M downstream probe, 0.5U/. mu.L Ampligase (Epicenter), 1U/. mu.L RiboLock RNase inhibitor (Fermantas), 50mM KCl and 20% formamide was added to the sample, and after incubation at 37 ℃ for 30 minutes, incubation was carried out at 45 ℃ for 45 minutes. Hybridizing a pair of ligation probes specifically complementary to the target sequence in the cDNA; each pair of connecting probes includes an upstream probe
(5'-GCCGGCTTCGCGGGCGACGATTCCTCTATGATTACTGACCTATGC-3', SEQ ID NO 02) and a downstream probe
(5'-GTCTATTTAGTGGAGCCTCTTCTTTACGGCGCCGGCATGTGCAAG-3', SEQ ID NO 03): the 3 'end of the upstream probe is provided with an upstream recognition sequence which is specifically complementary with the upstream sequence of the target sequence, and the 5' end is provided with an upstream loop-forming connecting template complementary sequence; the 5 'end of the downstream probe is provided with a downstream recognition sequence which is specifically complementary with the downstream sequence of the target sequence, and the 3' end is provided with a downstream loop-forming connecting template complementary sequence; the upstream sequence and the downstream sequence of the target sequence do not overlap with each other; specifically, the upstream probe and the downstream probe were subjected to T4PNK phosphorylation (a reaction mixture containing 2. mu.M of the probe, 1 XPNK buffer A (Fermentas), 1mM ATP, and 0.1 u/. mu. L T4 of polynucleotide kinase (Fermentas) was reacted at 37 ℃ for 10min, followed by further reaction at 65 ℃ for 10min, and then stored at-20 ℃ for future use). Connecting an upstream recognition sequence and a downstream recognition sequence which are specifically hybridized and complemented with a target sequence by Ampligase DNA ligase (the connection reaction condition is 37 ℃, 45 ℃ after 30 min), and forming a first sequence; after completion of the reaction, the reaction was washed three times with DEPC-PBS-Tween 20.
(2) Probe looping
To the sample was added 50. mu.L of ligation mixture containing Ampligase buffer (Epicenter) at a final concentration of 1X, 1. mu.M of the circular template oligo (5'-CTAAATAGACGCATAGGTCA-3', SEQ ID NO 04), 0.5U/. mu.L of Ampligase (Epicenter), 1U/. mu.L of RiboLock RNase inhibitor (Fermantas), 50mM KCl and 20% formamide, and the mixture was incubated at 37 ℃ for 30 minutes and 45 minutes at 45 ℃. Enabling a loop-forming connection template to be simultaneously and specifically hybridized and complemented with an upstream loop-forming connection template complementary sequence and a downstream loop-forming connection template complementary sequence on two sides of a first sequence, wherein the upstream loop-forming connection template complementary sequence and the downstream loop-forming connection template complementary sequence are not overlapped with each other relative to the loop-forming connection template; the Ampligase DNA ligase connects the upstream cyclization connecting template complementary sequence which is specifically hybridized and complemented with the cyclization connecting template with the downstream cyclization connecting template complementary sequence to obtain an annular template;
(3) rolling circle amplification
Performing rolling circle amplification on the annular template to obtain a rolling circle amplification product, specifically: washing with DEPC-PBS-Tween 20 three times, adding 50uLDEPC treated water to the sample, and adding rolling circle amplification reaction mixture containing Phi29 buffer (Fermantas) with final concentration of 1 × 1u/μ l Phi29 polymerase (Fermantas), 0.25mM dNTP, 0.2mg/ml BSA and 5% glycerol at 37 deg.C for 2-3h or overnight at room temperature;
(4) detection probe hybridization and image acquisition
Hybridizing the rolling circle amplification product with a detection probe (5'-TGCGTCTATTTAGTGGAGCC-3', SEQ ID NO 05, Cy3 marked at the 5' end), and finally carrying out cell nucleus staining by using DAPI for detection, wherein the detection is carried out specifically as follows: samples were washed three times with DEPC-PBS-Tween 20, added to 2 XSSC buffer containing 1. mu.M detection probe, 20% formamide, incubated at 37 ℃ for 30 minutes, and then subjected to gradient ethanol: 70%, 85%, 100% dehydration treatment, each for 5 min. And (5) air drying. Finally, 100ng/mL DAPI is added
Gold antipade mount (Fermantas) was incubated at room temperature for 10min before fluorescent microscopy and photographed. The results of the detection are shown in FIG. 3.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
<110> university of Chinese
<120> an in situ nucleic acid detection method
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gtctatttag tggagcctct tctttacggc gccggcatgt gcaag 45
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tgcgtctatt tagtggagcc 20
Claims (2)
1. An in situ nucleic acid detection method for non-diagnostic therapeutic purposes, characterized by: the method comprises the following steps:
(1) subjecting at least one pair of ligation probes to specific hybridization complementarity with a target sequence in a sample to be tested; each pair of ligation probes comprises an upstream probe and a downstream probe: the 3 'end of the upstream probe is provided with an upstream recognition sequence which is specifically complementary with the upstream sequence of the target sequence, and the 5' end is provided with an upstream loop-forming connecting template complementary sequence; the 5 'end of the downstream probe is provided with a downstream recognition sequence which is specifically complementary with the downstream sequence of the target sequence, and the 3' end is provided with a downstream loop-forming connecting template complementary sequence; the upstream sequence and the downstream sequence of the target sequence do not overlap with each other; the sample to be detected is cells in fixed cultured cells, tissues and tissue slices, the target sequence in the sample to be detected is cDNA, and the cDNA is formed by the in-situ reverse transcription of mRNA in the sample to be detected; the upstream probe and the downstream probe are subjected to phosphorylation treatment by T4 PNK;
(2) connecting an upstream recognition sequence and a downstream recognition sequence which are specifically hybridized and complemented with a target sequence in a sample to be detected by Ampligase DNA ligase to form at least one first sequence;
(3) enabling at least one looping connecting template to be simultaneously and specifically hybridized and complemented with an upstream looping connecting template complementary sequence and a downstream looping connecting template complementary sequence on two sides of at least one first sequence, wherein the upstream looping connecting template complementary sequence and the downstream looping connecting template complementary sequence are not overlapped with each other relative to the looping connecting template;
(4) connecting the upstream cyclization connecting template complementary sequence and the downstream cyclization connecting template complementary sequence which are specifically hybridized and complemented with the cyclization connecting template by Ampligase DNA ligase to obtain at least one circular template;
(5) performing rolling circle amplification on the at least one circular template under the action of Phi29 polymerase to obtain at least one rolling circle amplification product;
(6) and hybridizing the at least one rolling circle amplification product with at least one detection probe to detect.
2. The method of claim 1, wherein the in situ nucleic acid detection is performed by a method other than diagnostic therapy: the detection probe is modified with a fluorescent marker, a chromogenic marker, an enzyme marker, a radioactive marker, a magnetic marker or a luminous density marker.
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| CN109797201B (en) * | 2019-03-12 | 2023-01-24 | 厦门先能生物科技有限公司 | In-situ multiple nucleic acid detection method |
| CN110373451A (en) * | 2019-07-23 | 2019-10-25 | 华侨大学 | A kind of unicellular gene expression analysis method using the unicellular rna expression of flow cytomery |
| CN111534573A (en) * | 2020-05-26 | 2020-08-14 | 深圳百纳心致生命科学有限公司 | Probe composition and application thereof in nucleic acid in-situ detection |
| CN113337579B (en) * | 2021-05-13 | 2022-08-12 | 厦门先能生物科技有限公司 | Method for detecting the presence or level of one or more target nucleic acids in a sample |
| CN113564230B (en) * | 2021-07-27 | 2023-10-31 | 华侨大学 | In-situ detection method for circular RNA |
| CN113463203B (en) * | 2021-07-27 | 2024-08-30 | 厦门先能生物科技有限公司 | Construction method of in-situ sequencing library for realizing in-situ detection of multiple RNAs |
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