CN115627290A - Method for improving PCR detection sensitivity - Google Patents
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Abstract
The invention relates to a method for improving PCR detection sensitivity, which is based on the cooperative growth of lyotropic liquid crystal soft template auxiliary silver ions and combines an electrochemical deposition method to prepare a silver flower material; the method is characterized in that a high-concentration silver solution is prepared from a silver material and used as a carrier of a detection sample, and in a PCR tube, the silver solution is used for enhancing the fluorescence intensity generated by the detection sample while ensuring the purity of the detection sample. The silver flower material disclosed by the invention not only has excellent physicochemical characteristics such as large surface area and volume ratio, surface charge density and good thermal conductivity, but also has a plurality of tips, can further enhance a fluorescence signal, does not generate complex chemical reaction with a detection sample, does not participate in an amplification process, only simply enhances the fluorescence signal to ensure the purity of the detection sample, and can detect the enhanced fluorescence intensity even under the condition of less viruses, thereby avoiding the occurrence of false negative.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a method for improving PCR detection sensitivity.
Background
Various serious epidemics of human, animal and plant caused by biological factors (bacteria, deinsectia, viruses, etc.) such as ebola virus, zika virus, middle east respiratory syndrome coronavirus and novel coronavirus have attracted general attention of governments of various countries. Rapid diagnosis of viral infections in patients is particularly critical in order to effectively manage and control the progression of the disease.
The method for detecting the pathogens is to carry out microscopic examination by culturing the pathogens in vitro, but the method has the problems of complicated operation, long time consumption, higher technical requirements, difficult detection of some microorganisms which are difficult to culture and the like; the problems of missed detection, manual operation errors and the like are easily caused by long window period of enzyme-linked immunosorbent assay (ELISA) detection of pathogens by using an immunological method; in the method, DNA of a person to be detected is extracted and put into a reaction instrument for DNA in-vitro amplification, a probe containing fluorescein is cut off during the DNA amplification of the virus, fluorescence is generated under the action of exciting light, and the fluorescence generated by a sample to be detected is amplified repeatedly to determine whether the virus contains the pathogen or not. However, from the market feedback, the PCR detection technology needs to be carried out in a laboratory with a certain foundation, and the amplification period is long, so that the detection time is greatly delayed, and the real-time large-scale detection is very inconvenient; and if the patient is infected early and the virus content is low, the false negative condition is easy to occur. In response to the above problems, some external substances are usually added to improve the sensitivity, accuracy and specificity of PCR detection, and such substances are called PCR enhancers.
The currently studied PCR enhancers mainly include small molecule chemical enhancers and nanomaterial enhancers. The small molecule chemical enhancer used in the traditional method is combined with DNA, DNA polymerase or primer in a detection sample, so that the sensitivity and specificity of PCR detection are improved; due to the excellent physicochemical characteristics of the nano material such as large surface area and volume ratio, surface charge density, good thermal conductivity and the like, the nano material has wide application prospect for PCR detection which needs temperature control for reaction, and the method is called nano-polymerase chain reaction (nano-PCR). At present, the chemical reduction method is mostly adopted to prepare the metal nanoparticle solution in the aqueous solution, and the amplification process is accelerated due to the excellent heat transfer performance, so that the PCR detection sensitivity is improved.
Because the chemical PCR enhancer used at present is combined with DNA, DNA polymerase or primers in a detection sample, the fluorescence intensity emitted by pathogens during PCR detection can be enhanced, and the sensitivity is improved. However, the method involves the problem of mutual binding among multiple substances, has very strict requirements on PCR enhancers, and is still inefficient for large-scale sample detection. The metal nanoparticles prepared by the chemical reduction method can improve the detection sensitivity by virtue of excellent heat transfer performance of metal, can accelerate the amplification process, and can be adsorbed on a primer due to small size, participate in the amplification process and possibly influence the purity of a detected sample.
The cause is as follows:
currently, small molecule chemical enhancers in the market are mainly dimethyl sulfoxide (DMSO) and n, n, n-trimethylglycine (betaine) as PCR enhancers. The former is often used as part of the PCR standard amplification optimization, and the latter as an osmoprotectant, which prevents DNA polymerase denaturation. The PCR intensifier has the main action mechanism that 1) the function of in-vivo single-strand binding protein can be simulated in a certain mode and is combined with single-strand DNA, so that the mismatching rate between a primer and a template is reduced, and the accuracy is improved; 2) Can have strong interaction or combination with a DNA template or DNA polymerase, thereby improving the local concentration of the polymerase or the DNA template and improving the specificity and the detection efficiency of PCR to a certain extent; 3) The specific combination between the template and the primer is improved, and the detection error rate is reduced. Therefore, the enhancer needs to be combined with DNA, DNA polymerase or primers to play a role, so that the selection requirement on the enhancer is very strict, and the generated fluorescence intensity needs to reach the detection threshold value through the amplification times and cannot reach real-time large-scale detection.
The gold/silver nanoparticle solution prepared by the chemical reduction method is mixed with a detection sample, and the small-sized silver nanoparticles are adsorbed on the primers, so that the dissociation of PCR products in a denaturation step can be accelerated to improve the PCR efficiency due to good thermal dispersion, but the phase change influences the purity of the detection sample, and false negative can be generated when the pathogen content is low.
Aiming at the defects that the used PCR intensifier has higher cost and rigorous material selection, influences the purity of a detected sample, cannot realize real-time large-scale detection and the like. Because the PCR detection finally depends on detecting the fluorescent signal to determine the existence of the pathogen, if the fluorescent signal can be greatly enhanced under the condition of not reacting with any substance in the detection sample, the PCR amplification times can be reduced, the detection sensitivity is effectively improved, and the false negative probability can be reduced by the enhanced fluorescence for the virus and the sample with small content. Therefore, the invention provides a method for improving the PCR detection sensitivity.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a method for improving PCR detection sensitivity, wherein a silver flower structure prepared based on a lyotropic liquid crystal soft template and an electrochemical reaction directly enhances a fluorescence signal so as to achieve the purpose of PCR detection sensitivity enhancement.
In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:
a method for improving PCR detection sensitivity comprises the following steps:
based on the cooperative growth of the lyotropic liquid crystal soft template and the auxiliary silver ions, preparing a silver material by combining an electrochemical deposition method;
the method is characterized in that a high-concentration silver solution is prepared from a silver material and used as a carrier of a detection sample, and in a PCR tube, the silver solution is used for enhancing the fluorescence intensity generated by the detection sample while ensuring the purity of the detection sample.
Furthermore, the lyotropic liquid crystal is prepared by mixing a surfactant, oil and water, wherein the surfactant is connected with an oil phase and a water phase to form a water-in-oil stable inverse hexagonal phase to form the lyotropic liquid crystal, a silver plate is used as an anode, ITO glass is used as a cathode to prepare a liquid crystal box, the liquid crystal box is placed into the lyotropic liquid crystal to be used as a lyotropic liquid crystal soft template, direct current voltage is applied to the two electrodes to carry out electrochemical reaction, silver ions in the water phase are sealed in a cavity of the lyotropic liquid crystal soft template, the silver gradually enlarges along with the progress of deposition, the limitation of the lyotropic liquid crystal soft template is finally broken through, a flower-shaped structure is finally formed under the action of a self-assembly effect, and the prepared silver is separated from the cathode ITO glass to form an aqueous solution containing silver materials, namely a silver solution.
Further, during PCR detection, a sample to be detected, a probe containing fluorescein and a silver flower solution are placed in a PCR tube as a PCR reaction reagent, the PCR tube is placed in a sample groove to be amplified, the probe containing fluorescein is cut off by a primer in the amplification process, so that the probe is dissociated in the sample to be detected and is used for being excited by excitation light to generate a fluorescent signal, when a weak fluorescent signal meets the silver flower in the sample to be detected, the fluorescent signal is enhanced, the detection sensitivity is improved, the fluorescent signal is gradually increased along with the increase of amplification time to reach a detection threshold, the concentration of the detected nucleic acid is obtained according to the contrast of the intensity of the fluorescent signal at the moment and a relation curve of the standard concentration of the detected nucleic acid and the fluorescent intensity, and then the amplification times are divided, and the initial concentration of the sample is quantitatively obtained.
Further, the lyotropic liquid crystal employs a dinaphthosuccinate (AOT; 98 wt%) as a surfactant, paraxylene (99 wt%) as an oil phase, and a silver nitrate solution as an aqueous phase.
Further, the gap between the two poles of the liquid crystal cell and the outer cell wall is used as the cell thickness and is controlled by the plastic sheet.
The invention has the beneficial effects that:
1. the preparation cost is low:
chemical enhancers are used at higher cost due to their specificity, which requires fusion with specific DNA, DNA polymerases, or primers. The honeysuckle aqueous solution prepared by the method is simple to prepare and low in material cost.
2. Has higher PCR detection sensitivity:
the chemical reinforcing agent and the metal nano-particles are mixed with pathogens to react, so that the amplification process is accelerated, but the amplification is still carried out for multiple times when the fluorescence detection threshold is reached, and the sensitivity cannot be detected in real time on a large scale. The silver flower structure has excellent thermal conductivity, so that the reaction temperature can be quickly increased or decreased, and the reaction time is shortened; the fluorescence excited by the various tip structures of the honeysuckle is greatly enhanced, the fluorescence intensity is increased along with the increase of the concentration of the used honeysuckle solution, and the optimal concentration of the honeysuckle solution is selected to enable the fluorescence intensity to reach a detection threshold value more quickly, so that the PCR detection has higher sensitivity.
3. Does not affect the purity of the detection sample:
the chemical enhancer needs to be fused with specific DNA, DNA polymerase or primers to improve the PCR detection sensitivity, the metal nanoparticles prepared by the chemical reduction method need to adsorb the primers to accelerate the amplification period, and both the chemical enhancer and the primers participate in the reaction process of a detection sample in a PCR instrument and possibly influence the detection sample. The honeysuckle structure only enhances the fluorescence emitted during the amplification of the detection sample, does not participate in the amplification process, can well ensure the purity of the detection sample, and has higher detection accuracy.
4. Has wider application market:
the existing PCR intensifier needs to select a specific intensifier for mixed detection according to different pathogens, and the silver flower material intensification technology can be applied in a coating or integrated PCR tube form after being mature, so that the problem of separation of a PCR sample and a nano material can be solved, and the PCR intensifier can better circulate in the market. And the honeysuckle material only simply improves the fluorescence intensity, so that the silver fluorescent material has higher universality and is more favorable for popularization of PCR detection.
Drawings
FIG. 1 is a schematic diagram of the preparation of the silver flower material of the present invention: (a) the reaction device and its lyotropic liquid crystal phase; (b) self-assembly and co-growth of silver flowers;
FIG. 2 is a schematic diagram of the PCR reaction reagent according to the present invention;
FIG. 3 is a schematic diagram of the light path of the PCR reaction of the present invention;
fig. 4 is an SEM characterization of a silver substrate of the invention: (ii) (a) the prepared silver substrate; (b) position 1; (c) position 2; (d) position 3; (e) position 4; (f) position 5; (g) a single silver flower structure;
FIG. 5 is a graph showing the effect of the silver flower solution of the present invention on PCR fluorescence enhancement;
FIG. 6 is a graph showing the enhancement effect of different concentrations of silver flower solution on PCR fluorescence according to the present invention;
FIG. 7 is a graph showing the effect of the concentration of the silver flower solution of the present invention on the fluorescence intensity.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings in combination with embodiments.
A method for improving PCR detection sensitivity comprises the following steps:
based on the cooperative growth of the lyotropic liquid crystal soft template and the auxiliary silver ions, preparing a silver material by combining an electrochemical deposition method;
the method is characterized in that a high-concentration silver solution is prepared from a silver material and used as a carrier of a detection sample, and in a PCR tube, the silver solution is used for enhancing the fluorescence intensity generated by the detection sample while ensuring the purity of the detection sample.
The lyotropic liquid crystal is prepared by mixing a surfactant, oil and water, wherein the surfactant is connected with an oil phase and a water phase to form a water-in-oil stable inverse hexagonal phase to form lyotropic liquid crystal, a silver plate is used as an anode, ITO glass is used as a cathode to prepare a liquid crystal box, the liquid crystal box is placed into the lyotropic liquid crystal to be used as a lyotropic liquid crystal soft template, direct current voltage is applied to the two electrodes to carry out electrochemical reaction, silver ions in the water phase are sealed in a cavity of the lyotropic liquid crystal soft template, the silver flowers gradually grow along with the deposition, the limitation of the lyotropic liquid crystal soft template is finally broken through, a flower-shaped structure is finally formed under the action of a self-assembly effect, the preparation and reaction processes are shown in figure 1, the prepared silver flowers are separated from the cathode ITO glass, and an aqueous solution containing silver flower materials, namely a silver flower solution, is formed.
During PCR detection, as shown in FIG. 2, a sample to be detected, a probe containing fluorescein and a silver flower solution are placed in a PCR tube together as a PCR reaction reagent; as shown in fig. 3, the PCR tube is placed in the sample tank for amplification, and through amplification, the target gene or DNA fragment to be detected can be amplified to hundreds of thousands to millions of times within several hours, the primer cuts off the probe containing fluorescein during amplification, so that the probe is dissociated in the sample to be detected and is excited by the excitation light to generate a fluorescence signal, when the weak fluorescence signal encounters the honeysuckle in the sample to be detected, the fluorescence signal is enhanced, thereby greatly improving the detection sensitivity, shortening the detection time, and gradually increasing with the increase of the amplification time to reach the detection threshold, and the concentration of the detected nucleic acid is obtained according to the comparison between the intensity of the fluorescence signal at this time and the relationship curve between the standard concentration of the detected nucleic acid and the fluorescence intensity, and then divided by the amplification times to quantitatively obtain the initial concentration of the sample; meanwhile, the honeysuckle material can not participate in the whole amplification process, so that the purity of a sample to be detected is ensured, and the fluorescence signal generated in the amplification process is greatly enhanced, so that the detection threshold can be reached only by few amplification times, and the PCR detection sensitivity is greatly improved.
The lyotropic liquid crystal uses dinaphthosuccinate (AOT; 98 wt%) as a surfactant, p-xylene (99 wt%) as an oil phase, and a silver nitrate solution as an aqueous phase.
The gap between the two poles of the liquid crystal cell and the outer cell wall is used as the cell thickness and is controlled by the plastic sheet.
Example (b):
firstly, preparing lyotropic liquid crystal by using double sodium sulfosuccinate (AOT; 98 wt%), paraxylene (99 wt%) and 0.3M silver nitrate solution, wherein the ratio of the lyotropic liquid crystal is as follows: [ AOT ] < H2O ] =10, [ AOT ] < P-xylene ] =1.4;
stirring and standing for 2 hours to form lyotropic liquid crystal; then, a silver plate is used as an anode, ITO glass is used as a cathode to prepare a liquid crystal box, and the thickness of the liquid crystal box is controlled by a 0.7mm plastic sheet;
then, putting the liquid crystal box into a lyotropic liquid crystal, applying 5V direct current voltage to the two poles to perform electrochemical reaction, and stopping the reaction after 3 hours;
after the reaction is finished, taking out the liquid crystal box, immersing the liquid crystal box in absolute ethyl alcohol to separate the two polar plates, finally, putting the separated ITO glass into clean absolute ethyl alcohol, cleaning the residual lyotropic liquid crystal on the silver substrate by using an ultrasonic cleaning machine, and storing after the cleaning and drying are finished;
scanning characterization of the silver material is performed by using a thermal field emission Scanning Electron Microscope (SEM), as shown in fig. 4, a gray area in fig. 4 (a) is the prepared silver material, fig. 4 (b), (c), (d), (e), (f) respectively correspond to 5 different positions 1, 2, 3, 4, 5 in fig. 4 (a), it can be seen that the structure of the silver grown on the ITO glass is uniformly distributed, fig. 4 (g) is an enlarged single silver material, the diameter of the silver material is about 4 μm, the thickness of petals is about 100nm, and it can be seen that a plurality of tips beneficial to enhancing fluorescence signals exist on the surface of the silver.
In order to utilize the silver material grown on the ITO glass to perform fluorescence enhancement of PCR, the silver material is required to be stripped from the ITO glass and prepared into silver solution with different concentrations, rhodamine 6G (R6G) is used for replacing a detection sample in an experiment, the enhancement effect of the silver on the fluorescence intensity is verified, the silver solution and the R6G are mixed, the mixture is moved into a PCR tube through a liquid moving gun, and the PCR tube is placed into a PCR instrument for amplification and fluorescence intensity detection.
In order to compare the sensitivity effect of the silver flower material on the PCR, water and the silver flower solution are respectively added into the R6G solution with the same concentration, and then a PCR instrument is used for detection, and the result is shown in figure 5, and it can be seen that after the silver nano solution is added, the fluorescence signal of the PCR is obviously enhanced, and is improved by about 1 time.
Further exploring and comparing the fluorescence effect enhancement effect of the silver solution with different concentrations on PCR detection, further improving the PCR detection sensitivity, wherein the experimental result is shown in FIG. 6, compared with the R6G solution without silver, the R6G solution with the initial concentration is added, the fluorescence intensity is enhanced by about 2.72 times, the silver solution is enhanced by 3.42 times after 2 times of concentration, the silver solution is enhanced by 3.69 times after 3 times of concentration, and the relation between the silver solution concentration and the fluorescence intensity is obtained; as shown in fig. 7, the relationship is linear, so that the higher the concentration of the added silver solution is, the higher the fluorescence intensity enhancement effect is, and the higher the PCR detection sensitivity is, while ensuring that the detection sample is not affected, i.e., while no metal is precipitated.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A method for improving PCR detection sensitivity is characterized in that the method comprises the following steps:
based on the cooperative growth of the lyotropic liquid crystal soft template and the auxiliary silver ions, preparing a silver material by combining an electrochemical deposition method;
a high-concentration silver solution prepared from a silver material is used as a carrier of a detection sample, and in a PCR tube, the silver solution is used for enhancing the fluorescence intensity generated by the detection sample while ensuring the purity of the detection sample.
2. The method for improving the sensitivity of PCR detection according to claim 1, wherein the lyotropic liquid crystal is prepared by mixing a surfactant, oil and water, the surfactant is connected with the oil phase and the water phase to form a water-in-oil stable reversed hexagonal phase to form a lyotropic liquid crystal, a silver plate is used as an anode, ITO glass is used as a cathode to prepare a liquid crystal box, the liquid crystal box is placed in the lyotropic liquid crystal as a lyotropic liquid crystal soft template, a direct current voltage is applied to the two electrodes to carry out an electrochemical reaction, silver ions in the water phase are enclosed in a cavity of the lyotropic liquid crystal soft template, the silver gradually becomes larger along with the progress of deposition, the limitation of the lyotropic liquid crystal soft template is finally broken through, a flower-like structure is finally formed under the effect of self-assembly, and the prepared silver is separated from the cathode ITO glass to form an aqueous solution containing silver materials, namely a silver solution.
3. The method of claim 2, wherein during the PCR detection, the sample to be detected, the probe containing fluorescein and the silver flower solution are placed in the PCR tube as PCR reagents, the PCR tube is placed in the sample tank for amplification, the probe containing fluorescein is cut off by the primer during the amplification process, so that the probe is dissociated in the sample to be detected and is excited by the excitation light to generate a fluorescence signal, when the weak fluorescence signal encounters the silver flower in the sample to be detected, the fluorescence signal is enhanced, the detection sensitivity is improved, the fluorescence signal is gradually increased along with the increase of the amplification time to reach a detection threshold, the concentration of the detected nucleic acid is obtained according to the comparison between the intensity of the fluorescence signal and a relation curve between the standard concentration of the detected nucleic acid and the fluorescence intensity, and the initial concentration of the sample is quantitatively obtained by dividing the amplification times.
4. The method for improving the detection sensitivity of PCR according to claim 2 or 3, wherein the lyotropic liquid crystal comprises di-sodium sulfosuccinate (AOT; 98 wt%) as surfactant, p-xylene (99 wt%) as oil phase, and silver nitrate solution as water phase.
5. The method for improving the sensitivity of PCR detection according to claim 4, wherein the gap between the two poles of the liquid crystal cell and the outer wall of the liquid crystal cell is used as the cell thickness and is controlled by a plastic sheet.
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