A molecular beacon-g quadruplex optical sensor and its application in the detection of sv40 virus
An optical sensor and molecular beacon technology, applied in the field of biochemical analysis, can solve problems such as insufficient sensitivity, complex operation process, unsuitable for large-scale screening and detection at the grassroots level, and achieve good selectivity.
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Embodiment 1
[0071] Example 1 Detection of SV40 virus
[0072] (1) In 250μL of 2μmol / L molecular beacon buffer solution of PBS (20mM, pH 7.4), add 50μL of a series of different concentrations of target duplex DNA (Oligo-1 and Oligo-2) or test samples respectively , 50μL 5mmol / L arginine, 50μL 3μmol / L silver ion and 50μL 1mol / L potassium ion, at 25 ℃ for 1.5 hours;
[0073] (2) Add 50 μL of 40 μM NMM to the above solution, and incubate at 25°C for 15 minutes; that is, the final concentrations of arginine, silver ions and potassium ions in the reaction system are 0.5 mmol / L, 0.3 μmol / L and 100 mmol, respectively. / L;
[0074] Then, the mixed solution was transferred to a micro-quartz sample cell (3mm×10mm×47mm) to measure the fluorescence emission at a wavelength of 614nm; wherein, the fluorescence spectrum measurement parameters were set: excitation wavelength 399nm, slit width 5nm, emission wavelength scanning range 580 ~650nm.
[0075] The experimental schematic is as follows figure 1...
Embodiment 2
[0078] Embodiment 2 Detection of SV40 virus
[0079] (1) In 250μL of 2μmol / L molecular beacon buffer solution of PBS (20mM, pH 7.4), add 50μL of a series of different concentrations of target duplex DNA (Oligo-1 and Oligo-2) or test samples respectively , at 25°C for 1.5 hours;
[0080] (2) Add 50 μL of 40 μM NMM to the above solution, and incubate at 25°C for 15 minutes; then, transfer the mixture to a micro-quartz sample cell (3mm×10mm×47mm) to measure the fluorescence emission at a wavelength of 614nm; Spectral measurement parameter settings: excitation wavelength is 399 nm, slit width is 5 nm, and emission wavelength scanning range is 580-650 nm.
[0081] The sensor of this embodiment has the advantages of high sensitivity, good specificity and good selectivity for the detection of SV40 virus. When the concentration of SV40 virus double helix DNA sequence was in the range of 5-300 nmol / L, the fluorescence signal value increased with the increase of target strand DNA conc...
Embodiment 3
[0082] Example 3 Variation of fluorescence intensity with spermine concentration
[0083] 1. Method
[0084] On the basis of Example 1, the spermine concentration of the reaction system is used as a single factor variable, and ΔI is used as an indicator (ΔI is defined as the difference in fluorescence intensity, and the definition formula of ΔI is ΔI=I target -I blank , I target Indicates the presence of the fluorescent signal of the target strand, I blank Indicates the fluorescence signal without the addition of the target strand), to investigate the effect of different spermine concentrations on the detection effect of the SV40 virus-specific target duplex DNA sequence.
[0085] 2. Results
[0086] The experimental results are as figure 2 As shown, the change of spermine concentration significantly affects the intensity of the detected fluorescence intensity. When the spermine concentration is in the range of 0.1 to 0.5 mmol / L, the absorbance value increases with the i...
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