Real-time quantitative PCR detection method for aflatoxin B1
An aflatoxin, real-time quantitative technology, used in the determination/inspection of microorganisms, biochemical equipment and methods, etc.
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experiment example 1
[0048] Experimental example 1 Complementary single-stranded DNA and PCR primer specificity optimization experiment
[0049] The complementary single-stranded DNA immobilized on the surface of the PCR tube is used as a template for PCR amplification, and its concentration and the specificity of the PCR primers are very important; in order to improve the detection effect, the present invention optimizes the concentration of the complementary single-stranded DNA and the PCR detection primers.
[0050] The optimized experimental process is as follows: ABI7500 real-time PCR system is used for detection, and the 50 μL PCR reaction system consists of the following parts: concentration range 1×10 -4 5 μL of complementary single-stranded DNA to 10 nM, 10 μM upstream and downstream primers (SEQ ID No.3 and SEQ ID No.4) were added 2 μL, 25 μL Premix Ex (2×), 1 μL ROX Reference Dye II (50×) and 15 μL water. The real-time PCR cycle parameters were set as follows: pre-denaturation 30s95...
experiment example 2
[0055] Experimental example 2 Concentration optimization experiment of streptavidin, biotinylated aptamer and complementary single-stranded DNA
[0056] The concentration of streptavidin, biotinylated aptamer and complementary single-stranded DNA can greatly affect the detection performance of aptasensor. In order to obtain the best detection effect, the present invention optimizes the concentration of streptavidin, biotinylated aptamer and complementary single-stranded DNA.
[0057] The optimization experiment process is as follows: 1) The concentration of fixed complementary DNA is 10nM, the concentration of streptavidin and aptamer is optimized by analyzing the change of PCR amplification signal, 50μL of 0.8% glutaraldehyde solution is used to treat the PCR tube at 37°C for 5h . After washing with ultrapure water three times, add streptavidin dissolved in 0.01M carbonate buffer (concentrations are 2.5, 5, 10ng mL -1 )50μL and incubated for 2h (37℃), washed twice with PBST...
Embodiment 1
[0062] Example 1 Aptamer-based AFB 1 Establishment and method validation of real-time quantitative PCR detection method
[0063] 1. Immobilization of aptamers
[0064] Immobilization of aptamers and some modifications. In order to improve the adsorption effect, the PCR tube was treated with 50 μL of 0.8% glutaraldehyde solution at 37°C for 5h before use. After washing with ultrapure water for three times, add 50 μL of streptavidin dissolved in 0.01M carbonate buffer and incubate for 2 hours (37°C). : 1 (v / v) Mix well, add 50 μL of the mixture to each tube and incubate for 1 h (37°C). To remove unbound DNA fragments, wash the PCR three times with hybridization buffer to leave the aptamer and modified DNA on the surface of the PCR tube.
[0065] 2. RT-qPCR conditions
[0066] ABI7500 real-time PCR system was used for detection. The 50 μL PCR reaction system consisted of the following parts: 10 μM upstream and downstream primers were added 2 μL, 25 μL Premix Ex (2×), 1 μ...
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