Method for detecting nucleic acids by simultaneous isothermal amplification of nucleic acids and signal probe
A technology of isothermal amplification and signal probe, applied in the field of temperature amplification of nucleic acid and signal probe, can solve the problems of low specificity, high cost of RNA monomer and high cost
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Embodiment 1
[0071] Example 1: Isothermal amplification of DNA
[0072] Chlamydia trachomatis (ATCC VR-887) DNA was used as target nucleic acid. Genomic DNA using G-spin TM Genomic DNA extraction Kit (iNtRON Biotechnology, Cat.No.17121) is extracted from the Gram-negative bacteria Chlamydia trachomatis, and then amplified. For genomic DNA extraction, 500 mL of the bacterial suspension was centrifuged at 13,000 rpm for 1 min, and then the supernatant was removed, and 500 mL of PBS (pH 7.2) was added thereto, followed by centrifugation to remove the supernatant. Then suspend the cell pellet by adding 300ml of G-buffer solution containing RNase A and proteinase K, let it stand at 65°C for 15min, then add 250mL of binding buffer to it, mix well, and then bind the DNA to the spin column. After that, add 500 mL of Wash Buffer A to the spin column, centrifuge at 13,000 rpm for 1 min to wash, add 500 mL of Wash Buffer B to the spin column for centrifugation, then move the column to a 1.5 mL mic...
Embodiment 2
[0086] Example 2: DNA Detection by Enzyme Immunoassay
[0087] 170 ml of PBST binding buffer was added to the amplification product obtained in Example 1 to prepare a reaction mixture consisting of the following components: 136 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 0.05% Tween 20 , anti-F-HRP (PerkinElmer, horseradish peroxidase-conjugated anti-fluorescein antibody) diluted 1 / 7000. The reaction mixture was transferred to streptavidin-coated microplate wells (Roche) and reacted at 37°C and 200 rpm for 10. The supernatant of each well was removed, and 300 ml of PBST washing buffer was added to each well for washing, wherein the PBST washing buffer had the same composition as the above-mentioned binding buffer except that antibodies were removed therefrom. After washing, 200ml of HRP substrate, 3,3′,5,5′-tetramethylbenzidine (Bio-Rad, TMB) was added to each well, incubated in the dark for 5min to develop color, and then 100ml of 1N H2SO4 was added to stop reaction...
Embodiment 3
[0089] Example 3: DNA Detection by Lateral Flow Chromatography
[0090] 10 ml of colloidal gold solution (Chemicon) with a diameter of 40 nm was added to 100 mg of streptavidin (Sigma), vortexed for 2 min, and then reacted for 3 hr. Then, 1 mL of 1% BSA (dissolved in 2 mM borate) solution was added to the resulting mixture, centrifuged at 10,000 rpm for 15 min at 4°C, the supernatant was removed, and then 1 mL of 2 mM borate buffer was added to the supernatant The resultant was washed 3 times and then resuspended by adding 1% BSA (dissolved in 2 mM borate).
[0091] The gold conjugate solution having an absorbance of 10 at 520 nm was stored at 4°C and used by diluting to an appropriate ratio. On nitrocellulose membranes, fluorescein antibody (Chemicon) was coated as a test line and biotin-conjugated casein (Biofocus) was coated as a control line, respectively.
[0092] Add 60 mL of gold conjugate solution diluted 1:50 with running buffer (1x PBS, 1% Triton X-100, 0.6% BSA) t...
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