SARS-CoV-2 detection system and detection method based on hybridization chain reaction
A hybrid chain reaction, sars-cov-2 technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/inspection, etc., can solve cumbersome modification steps, delay detection time, increase detection cost, etc. problems, to achieve good stability, reduce the demand for complex equipment, and less interference factors
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Embodiment 1
[0058] The present invention selects two RNA gene fragments ORF1ab (SEQ ID No. 4) and N from the open reading frame and nucleocapsid according to the SARS-CoV-2 virus genome information (NCBI reference sequence: NC_045512.2) that has been published in the NCBI database (SEQ ID No. 5), and then designed a connector based on these two gene fragments, an initiator sequence Initiator and three sets of hybridization chain reaction hairpin probes, respectively H1 13 and H2 13 , H1 15 and H2 15 , H1 17 and H2 17 , and the specific sequence is shown in Table 1. It was then synthesized and purified by Sangon Bioengineering Co., Ltd. (Shanghai, China).
Embodiment 2
[0059] Example 2 Verification of DNA triplet formation
[0060] 1. Polyacrylamide gel electrophoresis
[0061] By mixing 6.25 mL of 40% acrylamide / bis-acrylamide solution (19:1), 5 mL of 1x TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0), 13.75 mL of ultrapure water, 180 μL of 0.1 g 10% polyacrylamide gel was prepared with 18 μL APS / mL APS and 18 μL TEMED. Take 6 μL of ORF1ab sequence, N sequence, connector sequence and ORF1ab+N+connector sequence, respectively, mix with 4 μL HEPES buffer (10 mM HEPES, 300 mM NaCl, pH 7.0) and 2 μL 6×loading Buffer, and inject 2 μL of the mixture into the gel. in the glue hole. The gel was run in 1×TBE buffer at 4°C and 150v for 5h, stained with 1×SYBR Gold for 30min, and then imaged with an imaging system. The results are as follows figure 2 shown.
[0062] Depend on figure 2 It can be seen that the connector hybridizes with the 3'-end domain of ORF1ab and the 5'-end domain of N to induce DNA triplet formation.
Embodiment 3
[0064] 1. Preparation and storage of DNA and RNA solutions
[0065] Concentrated DNA stocks of the sequences described in Table 1 were prepared in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and diluted to working concentration with HEPES buffer (10 mM HEPES, 300 mM NaCl, pH 7.0).
[0066] Concentration method: Dissolve the nucleic acid sequence in microliters of TE buffer solution marked on the tube, and measure the concentration of the concentrated solution by ultraviolet absorption spectroscopy.
[0067] Put H1 13 and H2 13 , H1 15 and H2 15 , H1 17 and H2 17 Heated at 95°C for 2 min and slowly cooled to room temperature. The annealed H1 13 and H2 13 , H1 15 and H2 15 , H1 17 and H2 17 Store at 4°C for later use.
[0068] 2. Selection of H1 and H2
[0069] H1 at a concentration of 400nM 13 and H2 13 , H1 15 and H2 15 , H1 17 and H2 17 In HEPES buffer, it was mixed with connector at a concentration of 100 nM, ORF1ab at a concentration of 100 nM, and N a...
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