Universal probe for detecting nucleic acid based on catalytic hairpin self-assembly isothermal amplification technology and application of universal probe
A hairpin probe and universal probe technology, applied in the field of molecular biology, can solve the problems of cumbersome and time-consuming, unsuitable for high-throughput screening, etc., and achieve the effect of low cost, low detection limit and good application prospects
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Embodiment 1
[0085] Example 1 Design, Feasibility Verification and Measurement Condition Optimization of CHA Detection System
[0086] 1. Start the hairpin H0 in the CHA detection system (H0 651 , H0 1246 , H0 39 ), hairpin probe H1, and hairpin probe H2 (the hairpin probe H1 is marked with Cy3, and the hairpin probe H2 is marked with Cy5) sequences are shown in Table 1.
[0087] Table 1 Dynamic hairpin H0 (H0 651 , H0 1246 , H0 39 ), the nucleotide sequence list of hairpin probe H1 and hairpin probe H2
[0088]
[0089] Note: H0 651 , H0 1246 , H0 39 The part in italics is the same sequence (SEQ ID NO.17), the stem part (stem part) is underlined, and both Cy3 and Cy5 are modified on the T base.
[0090]2. Electrophoresis experiment and fluorescence experiment to verify the feasibility
[0091] (1) Electrophoresis experiment
[0092] Initiate hairpin H0 in the CHA detection system (H0 651 , H0 1246 , H0 39 ), hairpin probe H1, and hairpin probe H2 for non-denaturing gel el...
Embodiment 2
[0123] Example 2 CHA detection system applied to exosome detection
[0124] In 200μL EP tubes, add the final concentration of 0, 1×10 6 pcs / μL, 5×10 6 pcs / μL, 1×10 7 pcs / μL, 5×10 7 pcs / μL, 1×10 8 cells / μL and 2×10 8 MCF-7 exosomes / μL (obtained by ultra-high-speed centrifugation from MCF-7 cell culture supernatant), add Triton X-100 with a final concentration of 1% and proteinase K with a final concentration of 84.5 μg / mL Incubate at 37°C for 30 min, then add H0 at a final concentration of 30 nM (H0 651 (The number of bases in the stem part is 10) / H0 1246 (the number of bases in the stem part is 9) / H0 39 (the number of bases in the stem part is 12)), H1 at a final concentration of 50nM, H2 at a final concentration of 100nM, and NaCl at a final concentration of 200mM, supplemented with PBS to a total volume of 100μL, and incubated at 45°C in the dark After 3 hours, use a fluorescence spectrophotometer to measure the fluorescence values at 564nm (FD) and 666nm (FA) with...
Embodiment 3
[0128] Example 3 CHA detection system is applied to the detection of clinical plasma samples
[0129] A number of plasma samples from breast cancer patients and healthy controls were collected from the Laboratory Department of Nanfang Hospital, Southern Medical University, respectively, and 100 μL was used for pretreatment: first, proteinase K (final concentration: 65 μg / mL) was added and incubated at 37°C for 30 minutes to destroy For free proteins in plasma, add protease inhibitor PMSF (final concentration: 5mM) and incubate at room temperature for 20min to terminate excess proteinase K, then add RNase A (final concentration: 4U / mL) and incubate at 37°C for 30min to destroy plasma exosomes Free RNA, then add SDS (final concentration: 0.2%) to suppress excess RNase A, then add final concentration of 1% Triton X-100 and proteinase K (final concentration: 84μg / mL) and incubate at 37°C for 40min, then pass through 2500 Centrifuge at ×g for 5 min, take 95 μL supernatant and add H...
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