Quantitative PCR detection method based on gold nanoparticles
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A technology of gold nanoparticles and detection methods, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. The success rate is not ideal and other problems, to achieve the effect of rapid detection, good versatility, and simple operation
Active Publication Date: 2018-07-31
SUN YAT SEN UNIV
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[0005] At present, there are mainly two ways to combine PCR technology with AuNPs probes. One is to use asymmetric PCR technology to generate single-stranded DNA products, and then hybridize with DNA modified on AuNPs probes; The concentration ratio of the downstream primers is difficult to optimize, and its success rate is often not ideal; the other is to prepare two AuNPs probes modified with different primers, and then perform PCR reactions, but due to the steric hindrance of the DNA modified on the surface of AuNPs , hindering the progress of the PCR reaction and reducing its amplification efficiency
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Embodiment 1
[0065] 1. Polymerase chain reaction based on special structural primers:
[0066] Design amplification primers with a hairpin structure, and introduce a hexaethylene glycol interarm (Spacer 18) into the loop. The sequences of the upstream and downstream primers are:
[0067] Upstream primer: 5’-GGG AGA GAA GAACT spacer18AG TTC TTC TCT CCC GAC AGG CCCGAA GGA ATA GA-3’
[0069] Carry out PCR reaction in a system containing template DNA (sequence is 5'-GAC AGG CCC GAA GGA ATA GAA GAA GAA GGT GGAGAG AGAG-3'), a pair of amplification primers, Taq DNA polymerase and dNTP to obtain bands at both ends PCR products with single-stranded DNA fragments.
[0070] The total volume of the PCR reaction solution is 50 μL, including 5 μL of 10× PCR buffer, 1 μL of 10 mM dNTP, 3 μL of 25 mM MgCl 2 , 0.5 μL 5U / μL Taq DNA polymerase, 10 μM / L upstream and downstream primers 2 μL each, 2 μL te...
Embodiment 2
[0087] 1. Polymerase chain reaction based on special structural primers:
[0088] Amplification primers with hairpin structure were designed, and a three-polyethylene glycol interarm (Spacer 9) was introduced into the loop, and the sequences of the upstream and downstream primers were:
[0091] PCR reaction was carried out in a system containing template DNA (sequence 5'-CTT CTC TTT GAT GTC ACG CAT ATG GAATCC TGT GGCATC-3', a pair of amplification primers, Taq DNA polymerase and dNTP to obtain PCR products of stranded DNA fragments.
[0092] The total volume of the PCR reaction solution is 50 μL, including 5 μL of 10× PCR buffer, 1 μL of 10 mM dNTP, 3 μL of 25 mM MgCl 2, 0.5 μL 5U / μL Taq DNA polymerase, 10 μM / L upstream and downstream primers 2 μL each, 2 μL template DNA o...
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Abstract
The invention provides a design method of amplification primers. The design method is characterized in that the amplification primers adopting hairpin structures are designed and single-stranded DNA fragments are formed at the two ends of an amplification product. The invention provides two gold nanoparticle probes; DNA sequences modified by the probes are complementary to the single-stranded DNAfragments at the two ends of a PCR product of DNA to be detected. The invention provides a quantitative PCR detection method based on gold nanoparticles. The quantitative PCR detection method comprises the following steps: (1), selecting a DNA sequence to be detected, and designing a pair of specially-structured amplification primers, wherein the pair of specially-structured amplification primersare designed to be the hairpin structures; (2), performing a PCR reaction in a system containing a DNA template to be detected, the pair of specially-structured amplification primers, Taq DNA polymerase and dNTP to obtain the PCR product; (3), preparing DNA-modified gold nanoparticle probes; (4), adding the probes into the PCR product, and hybridizing with the PCR product; (5), measuring the change of the absorbance or a dynamic light scattering signal of the gold nanoparticles. With the quantitative PCR detection method, the operation is simple, the PCR product can be rapidly detected, and the sensitivity is high.
Description
technical field [0001] The invention relates to a polymerase chain reaction (Polymerase Chain Reaction, PCR) detection method, which belongs to the technical field of biological detection, in particular to a quantitative PCR detection method based on gold nanoparticles. Background technique [0002] PCR is a technique for enzymatically amplifying DNA fragments in vitro and is the most commonly used method for detecting small amounts of target DNA molecules. However, the post-processing of PCR products by gel electrophoresis is laborious and time-consuming. [0003] Real-time fluorescent PCR (Real-time PCR) has made a major improvement on the traditional PCR technology, which can quantitatively detect target DNA molecules while amplifying. However, real-time fluorescent quantitative PCR instruments are relatively expensive and complicated, and their application in remote areas of underdeveloped countries will be limited. With this in mind, it is still necessary to develop n...
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