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Double block molecular probe and rapid nucleic acid detection method thereof

A molecular probe and diblock technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of time-consuming and increased detection costs, and achieve low preparation costs and detection The process is convenient and the effect of good specificity

Active Publication Date: 2016-03-16
GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method of probe preparation is usually time-consuming (20-40 hours)
In addition, probe modification with sulfhydryl groups also increases the cost of detection

Method used

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  • Double block molecular probe and rapid nucleic acid detection method thereof
  • Double block molecular probe and rapid nucleic acid detection method thereof
  • Double block molecular probe and rapid nucleic acid detection method thereof

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Experimental program
Comparison scheme
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preparation example Construction

[0038] 1. Preparation of diblock probes

[0039] (1) Design, synthesis and purification of diblock probes:

[0040] Design three diblock molecular probes: diblock molecular probe 1, diblock molecular probe 2 and diblock molecular probe 3; the diblock molecular probe 1 is sequentially from 5' to 3' Including: a promoter region, a hairpin structure region and a polyA region; wherein, the promoter region of the double-block molecular probe 1 is complementary to the partial sequence of the target DNA, and the promoter region of the double-block molecular probe 2 is complementary to the partial sequence of the double-block molecular probe The partial sequence of the hairpin structure region of 1 is complementary, the promoter region of diblock molecular probe 3 is complementary to the partial sequence of the hairpin structure region of diblock molecular probe 2, and the hairpin structure of the three diblock molecular probes After the region is opened, three double-block molecular...

Embodiment 1

[0053] 1. Preparation of diblock probes

[0054] (1) Design and synthesis of diblock probes

[0055] Design a diblock probe based on the target DNA sequence. The nucleic acid sequence of the target DNA is as follows:

[0056] y*b*x*a*

[0057] 5'-GCACTA-CTCCCT-AACATC-TCAAGC-3'(SEQ ID NO.1)

[0058] For the designed diblock probe, use NUPACK software to analyze the free energy of its two-dimensional structure. If the free energy is negative, the probe is feasible. The designed double-block probes were synthesized by a nucleic acid synthesis company, and the synthesized probes were purified by high-performance liquid phase, and mass spectrometry was used to detect whether the synthesized probes were consistent with the design. The successfully synthesized probes were freeze-dried and stored at -20°C. The specific sequence is as follows:

[0059] DHP1:

[0060] axbyz*c*y*b*x*

[0061] 5'-GCTTGA-GATGTT-AGGGAG-TAGTGC-TCCAAT-CACAAC-GCACTA-CTCCCT-AACATC-AAAAAAAAAAAAAAAA-3' (SE...

Embodiment 2

[0091] In order to further confirm that the method of the present invention is applicable to the bacterial nucleic acid detection process, according to the specific nucleic acid sequence of Shewanella outer membrane cytochrome c protein: 5'-TGGTCA-ATGTGT-TCTAGC-GAAGGT-3' (SEQ ID NO.5), design Three new diblock probes were developed. The specific sequence is as follows:

[0092] DHP1*:

[0093] 5'-ACCTTC-GCTAGA-ACACAT-TGACCA-TCCAAT-CACAAC-TGGTCA-ATGTGT-TCTAGC-AAAAAAAAAAAAAAAA-3' (SEQ ID NO. 6);

[0094] DHP2*:

[0095] 5'-ACACAT-TGACCA-GTTGTG-ATTGGA-ACACAT-GAAGGT-TCCAAT-CACAAC-TGGTCA-AAAAAAAAAAAAAAAA-3' (SEQ ID NO. 7);

[0096] DHP3*:

[0097]5'-GTTGTG-ATTGGA-ACCTTC-ATGTGT-TGGTCA-ATGTGT-TCTAGC-GAAGGT-TCCAAT-AAAAAAAAAAAAAAAA-3' (SEQ ID NO. 8).

[0098] The test results show (such as Figure 8 shown), when there is a bacteria-specific probe sequence, the color changes significantly, which illustrates the universality of the design of the double-block molecular probe and its...

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Abstract

The invention discloses a double block molecular probe and a rapid nucleic acid detection method thereof. Target nucleic acid sequences and initiating zones of the tail ends of the double block molecular probes interact mutually, strand displacement reaction is triggered, and hairpin structures of the double block molecular probes are opened sequentially and combined with target DNA, so that a lot of Y-type nucleic acid structures with three polyA tails can be generated. The polyA tails of the Y-type nucleic acid structures can be combined with a plurality of nanogold respectively, so that colors of the nanogold can be changed from red into blue, the detection result can be observed by naked eyes, and the nucleic acid concentration and the color change degree are in direct correlation. According to the double block molecular probe and the rapid nucleic acid detection method thereof, the operation is easy, protease and marking are not required, the reaction condition is mild, the detection limit to nucleic acid is 0.01 pM, single nucleic acid mutation can be distinguished, and the detection result can be used for quantitative analysis and nucleic acid rapid spot detection.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection, and relates to a diblock molecular probe and a method for rapidly detecting nucleic acid. Background technique [0002] Nucleic acid rapid detection technology has broad application prospects in the fields of medical diagnosis, food safety and environmental monitoring. Current nucleic acid detection methods mainly use proteases (such as nucleic acid polymerases, ligases, and exonucleases) as signal amplification tools. Although these methods can achieve lower detection limits, the operations of these methods are relatively complicated, time-consuming, labor-intensive, and relatively high in cost. Catalytic hairpin assembly is an enzyme-free signal amplification strategy that is often used to enhance nucleic acid hybridization signals. Catalytic hairpin assembly utilizes metastable hairpin probes to assemble Y-shaped nucleic acid structures. It can encode the information of the function an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 温俊林周顺桂陈俊华庄莉
Owner GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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