Application of a Fluorescent Probe with Dual Fluorescent Emissions in Detecting RNA

A fluorescent probe and fluorescent emission technology, applied in the application field of fluorescent probes and RNA detection, can solve the problems of low detection efficiency, low accuracy, low RNA detection specificity, etc., and achieve good linear correlation, The effect of specific detection and wide detection range

Active Publication Date: 2021-12-03
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
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Problems solved by technology

The intensity of the green fluorescence peak at 540nm is closely related to the double helix structure of double-stranded (deoxy) ribonucleotides, and can be used to detect the pairing situation of double-stranded (deoxy) ribonucleotides, and then detect the RNA and its mutations to be detected. This solves the technical problems of low specificity, low accuracy and low detection efficiency of RNA detection in the prior art

Method used

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  • Application of a Fluorescent Probe with Dual Fluorescent Emissions in Detecting RNA
  • Application of a Fluorescent Probe with Dual Fluorescent Emissions in Detecting RNA
  • Application of a Fluorescent Probe with Dual Fluorescent Emissions in Detecting RNA

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1: The ability of fluorescent probe TPBT to distinguish single-stranded RNA and double-stranded RNA

[0047] figure 2 Ribonucleotide A in different concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0 μM is added to SYBR Green I aqueous solution 50 with U 50 The composition of the double helix structure (A+U) 50 The fluorescence titration spectrum. image 3 Add 0, 0.2, 0.4, 0.6, 0.8μM different concentrations of single-stranded ribonucleotide A to the aqueous solution of SYBR Green I 50 Fluorescence titration spectra. Figure 4 It is in 10μM TPBT and excess polyanionic heparin sodium (200-2000μg·mL -1 ) to the aqueous solution composed of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0μM ribonucleotide A in different concentrations 50 with U 50 The composition of the double helix structure (A+U) 50 Fluorescence titration spectra. Figure 5 10μM TPBT with excess polyanion sodium heparin (200-2000μg·mL -1 ) to the aqueous solutio...

Embodiment 2

[0050] Embodiment 2: TPBT detects HIV RNA fragment and single base mutation thereof

[0051] 200 μg·mL was added dropwise to an aqueous solution of TPBT at a concentration of 10 μM -1 Heparin sodium, so that its 640 nm fluorescence emission intensity reaches balance, and the detection reagent of TPBT and heparin sodium is formed for use. SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 are HIV RNA, HIV RNA with single nucleotide mutation (mis HIV) and non-target RNA (nonHIV) and probe single-stranded DNA base sequence. Preparation of RNA-DNA double helix structure: Dissolve single-stranded probe DNA and RNA to be tested in ultrapure water to prepare a 0.5mM mother solution, mix probe DNA with target HIV RNA, single-base mutant mis HIV RNA and non- The target non-HIV RNA was mixed at 37°C and 225rad / min for 5-10min to obtain the corresponding RNA-DNA pair. Then the corresponding RNA-DNA pairs were titrated into the detection reagents of TPBT and sodium heparin respectiv...

Embodiment 3

[0053] Embodiment 3: Commercial dye SYBR Green I detects HIV RNA fragment and single base mutation thereof

[0054] Figure 10-13 In embodiment 3, commercial dye SYBR Green I detects the experiment of target RNA and single base mutation thereof. The DMSO mother solution of SYBR Green I at 10000× was diluted 10000 times with ultrapure water for use as a detection reagent. The preparation of the RNA-DNA double helix structure is the same as in the above-mentioned Example 3, and then the corresponding RNA-DNA pairs are respectively titrated into the detection reagent of SYBR GreenI, and the fluorescence intensity thereof is detected. Figure 10 It is the fluorescence spectrum of the RNA-DNA paired with HIV RNA and probe DNA in the detection reagent of SYBR Green I. Figure 11 It is the fluorescence spectrum of the RNA-DNA paired with the probe DNA titrated mis HIV RNA in the detection reagent of SYBR Green I. Figure 12 It is the fluorescence spectrum of the RNA-DNA paired wi...

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Abstract

The invention relates to the application of a fluorescent probe with double fluorescence emission in detecting RNA, and belongs to the technical field of molecular detection. When the fluorescent probe interacts with single-stranded (deoxy) ribonucleotides, it will only emit red fluorescence at around 640nm. When combined with double-stranded (deoxy) ribonucleotides in a double helix structure, a new Green fluorescence peak around 540nm. The green fluorescent peak at 540nm is closely related to the double helix structure of double-stranded (deoxy)ribonucleotides, and the fluorescent peak at 540nm can be used to specifically detect the existence of double-stranded (deoxy)ribonucleic acid. Utilizing this property of the fluorescent dye, a new application for rapid detection of RNA and RNA base mutations has been developed, which has a wider detection range and better linear correlation than the commercial dye SYBR Green I.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and more specifically relates to the application of a fluorescent probe with dual fluorescence emission in the detection of RNA. Background technique [0002] A major bottleneck affecting the prevention and control of the novel coronavirus is the diagnosis of patients with potential new crown infection. Both the production capacity of the kit and the time required for diagnosis are difficult to meet the needs of rapid detection of viral infection and effective isolation of viral infection in a short period of time. The core method for confirming cases is nucleic acid testing, but at present, nucleic acid testing is limited by cost and time, and the accuracy rate is not high, and false positive and false negative results often occur, thereby delaying the illness. Therefore, improving the speed and accuracy of nucleic acid detection is crucial to enhancing the prevention and control of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6816
CPCC12Q1/6816C12Q2563/107C12Q2527/125
Inventor 罗亮高玉婷孟凡玲何珍艳
Owner HUAZHONG UNIV OF SCI & TECH
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