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A constant temperature exponential amplification technique based on triple amplification reaction cascade and its application in microRNA detection

A technology of constant temperature index and constant temperature index amplification, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of high false positive rate, high detection background signal, complex primer design, etc., to reduce the system Effectiveness of background signal, improving assay specificity, avoiding false positive test results

Active Publication Date: 2018-08-14
SUN YAT SEN UNIV
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of high detection cost, complex primer design and high false positive rate caused by relying on PCR amplification technology in conventional nucleic acid analysis, and solve the limitations of high background signal and long analysis time of existing constant temperature target sequence cycle technology, realize Efficient, rapid and highly sensitive nucleic acid constant temperature analysis purposes

Method used

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  • A constant temperature exponential amplification technique based on triple amplification reaction cascade and its application in microRNA detection
  • A constant temperature exponential amplification technique based on triple amplification reaction cascade and its application in microRNA detection
  • A constant temperature exponential amplification technique based on triple amplification reaction cascade and its application in microRNA detection

Examples

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Effect test

Embodiment 1

[0059] Example 1 The composition of the constant temperature exponential amplification kit based on triple amplification reaction series for detecting miR-27a:

[0060] (1) Hairpin probe:

[0061] 5' End Neck Ring 3' End Neck

[0062] 5'-TTGACTC--GCGGAACTTAGCCACTGTGAA-- GAGTCAA--AT -3' (SEQ ID NO.1)

[0063] Thermostatic exponential amplification of template regions

[0064] The hairpin probe includes a 5' end neck, a loop, and a 3' end neck, and the 3' end neck extends at least 2nt to form an amplification fulcrum; the 3' end neck and the amplification fulcrum form a constant temperature exponential amplification template region, the The stretch sequence is likewise a nickase recognition sequence.

[0065] (2) Constant temperature exponential amplification primers:

[0066] 5'-TTGTTATCACCTATG -- ATT TGACTC-3' (SEQ ID NO.2)

[0067] 5' end sequence 3' end sequence

[0068] The constant temperature index amplification primer includes a 5' end sequence and a 3' end s...

Embodiment 2

[0082] Example 2 Establishment and specificity verification of a constant temperature exponential amplification detection method based on triple amplification reaction series for miR-27a

[0083] Prepare three reaction solutions A, B, and C on ice:

[0084] A reaction solution: 0.1 μM hairpin probe, 250 μM dNTP, 40 nM constant temperature exponential amplification primer, 0.8 U μL -1 RNase inhibitor and DEPC-treated water to a total volume of 10 µL.

[0085] B reaction solution: miRNA to be tested, 0.5×Nt.BstNBI buffer, 1×ThermoPol buffer, 0.4U·μL - 1 Nt.BstNBI nickase, 0.32U·μL -1 Bst DNA polymerase and DEPC-treated water to a total volume of 15 µL.

[0086] C reaction solution: 2mM Mg 2+ , 2 μM DNase substrate, 1 μM DNase and DEPC-treated water in a total volume of 5 μL.

[0087] Solution A was first incubated at 95°C for 5 minutes to denature the hairpin probes and bound primers, and then cooled to 55°C. Add solution B and mix, incubate at 55°C for 60 minutes and the...

Embodiment 3

[0090] Example 3 Optimization of the Constant Temperature Exponential Amplification Detection Method Based on Triple Amplification Reaction Series

[0091] The amplification efficiency of this method is affected by factors such as magnesium ions, incubation time and temperature, and the concentration of the working buffer. By optimizing, such as Figure 6 As shown, the best experimental conditions are obtained: Mg 2+ The concentration is 2mM; the incubation time is 60min; the incubation temperature is 55°C; the buffer system is ThermoPol buffer:Nt.BstNBI buffer=1:1.

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Abstract

The invention discloses a constant temperature index amplification technology based on triple amplification reaction connection in series and an application of the constant temperature index amplification technology in microRNA detection. According to the invention, a special neck ring structure is designed as a medium body, a constant temperature index amplification reaction is triggered, target sequence cycle is fromed under strand displacement reaction effect of polymerase, a lot of short chain DNA fragments are generated through amplification in short time, deoxyribozyme is activated, a fluorescence substrate is cut, target sequence cycle is formed, constant temperature index amplification reaction is carried out, series connection of a triple cycle amplification process of a deoxyribozyme catalytic reaction is carried out, then a rapid constant temperature index amplification technology with high efficiency is established, and the constant temperature index amplification technology can be used for microRNA ultra-sensitive fluorescence detection.

Description

technical field [0001] The invention belongs to the field of molecular detection, and in particular relates to a constant temperature exponential amplification technology based on triple amplification reaction series and its application in microRNA detection. Background technique [0002] MicroRNA (miRNA) is a non-coding single-stranded small molecule RNA with a length of 18-23 nucleotides, which widely exists in the cells of viruses, plants and higher mammals. It plays an important regulatory role in the process of life activities such as death and fat metabolism. MiRNA mutation or ectopic expression is associated with a variety of human cancers, can function as tumor suppressor genes or oncogenes, and can play an important role in the diagnosis and treatment of cancer. Therefore, the establishment of a rapid, accurate, highly selective and sensitive miRNA detection method is of great significance for in-depth research on the function and regulation of miRNA, as well as th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2525/301C12Q2521/345C12Q2525/207
Inventor 戴宗周雪晴邹小勇
Owner SUN YAT SEN UNIV
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