Zika virus nucleic acid detection method based on the principle of electrochemiluminescence amplification
A Zika virus and detection method technology, which is applied in the field of Zika virus nucleic acid detection based on the principle of electrochemiluminescence amplification, can solve the problems of cumbersome detection process, inability to realize Zika virus detection mode, and high price.
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Embodiment 1
[0110] Example 1 Technical route of Zika virus detection
[0111] (1) Zika virus nucleic acid extraction
[0112] ① Collect certain tissue or cell samples. The tissue and cell samples must be samples infected with Zika virus. The weight of a single extraction tissue should be less than 0.1g, and a single cell sample should not exceed 10 7 Cells, avoid too many samples to cause cell lysis ineffective;
[0113] ②Sample pretreatment:
[0114] a. Tissue samples
[0115] Grind the tissue into powder in liquid nitrogen, add 1mL sample protector, shake 3 to 5 times with a shaker (1 to 2 seconds each time), stand still for 30 minutes, remove the RNase in the cells, centrifuge for 30 minutes, and remove clear;
[0116] b. Monolayer adherent cell sample
[0117] Remove the liquid medium in the culture plate, add trypsin to the culture plate, digest the cells for 1 min, add medium to terminate the digestion, collect the cells by centrifugation and discard the supernatant, add 1 mL of sample protect...
Embodiment 2
[0148] Example 2 Probe characterization and verification of detection principle
[0149] In order to verify the feasibility of the probe construction of the present invention, the inventors characterized the probe labeling and DNA connection (experimental results are as follows: image 3 Shown in A and B). The inventor first detected the excitation and emission spectra of the terpyridine ruthenium polymer ( image 3 A), the experimental results show that the maximum absorption peak of the terpyridine ruthenium polymer appears at 460nm, and the emission peak is at 650nm, which is consistent with a single terpyridine ruthenium molecule. At the same time, the inventors characterized the product of the polymer connected to DNA nucleic acid, and the experimental results are as follows image 3 As shown in B, the single terpyridine ruthenium polymer molecule has an absorption peak at 460 nm. When the terpyridine ruthenium polymer and DNA probe are connected to construct an electrochemil...
Embodiment 3
[0151] Example 3 Optimization of detection conditions and performance evaluation
[0152] The key point of the probe construction system of the present invention lies in the ratio and purification of the terpyridine ruthenium polymer and the DNA recognition domain during the construction of the polymer probe. The inventors set different molecular ratios and purified the product by ultrafiltration. .
[0153] First of all, the inventors set different molecular ratio polymers, polymer: DNA recognition domain = 1:1, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1: 70, and in the case of sufficient reaction time, the electrochemiluminescence intensity of the probe after capturing the target nucleic acid was compared (experimental results such as Figure 4 As shown in A), it increases with the increase of the concentration of the DNA recognition domain, and reaches the plateau when the polymer:DNA recognition domain is 1:50. Therefore, the present invention takes polymer:DNA recognition domain=1...
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Abstract
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