CN110779970B - Electrochemical detection method for chicken infectious bronchitis virus H120 strain - Google Patents
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Abstract
The invention belongs to the technical field of analytical chemistry, and relates to an electrochemical detection method and application of an infectious bronchitis virus H120 strain. The method comprises the steps of equivalently converting RNA of an infectious bronchitis virus H120 strain into DNA (deoxyribonucleic acid) capable of specifically identifying the RNA of the H120 strain, namely H120-target, by annealing, enzyme digestion with S1nuclease (S1) and high-temperature heating, fixing a sulfydryl modified nucleic acid probe 2 on the surface of a gold electrode to incubate with the H120-target in a self-assembly mode, introducing a nucleic acid probe 1 modified gold nanoparticle capable of being specifically combined with the H120-target to adsorb an electric signal molecule ruthenium hexamine (RuHex) for generation and signal amplification, finally carrying out electrochemical detection on the adsorbed RuHex by using a linear voltammetry method, obtaining a linear equation by drawing a standard relation curve between the concentration of the H120 strain and an electric signal, and calculating the content of the H120 strain in an actual sample according to the size of the detected electric signal. The method has the advantages of low cost, high sensitivity and strong specificity.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and relates to an electrochemical method for detecting infectious bronchitis virus H120 strain.
Background
Infectious Bronchitis is an acute and highly contagious disease caused by Infectious Bronchitis Virus (IBV) of coronavirus, and causes huge economic loss to the chicken industry worldwide. IBV is a mononegavirale virus, the viral gene can be mutated by point mutation and recombination. IBV has more serotypes, and no cross reaction or little cross reaction among different serotypes, thereby increasing the difficulty of immunoprophylaxis. Therefore, it is of great practical significance to establish an effective method for detecting different strains of IBV to assist in the selection of a suitable vaccine.
Nanotechnology is a hotspot in the field of current material research, and gold nanoparticles are one of the most stable nanoparticles in metal nanoparticles and have the characteristics of good stability, small-size effect, surface effect, optical effect, good biocompatibility and the like. Based on the self-assembly of gold and sulfydryl, the gold nanoparticles and a sulfydryl modified specific DNA sequence form a probe, or a DNA modified electrode is prepared by self-assembling sulfydryl on the surface of gold, and the DNA modified electrode plays an important role in the research fields of biological medicine, gene therapy, clinical examination and the like.
The current methods for detecting viruses are immunoprecipitation, fluorescence quantitative PCR, enzyme linked immunosorbent assay, etc. These detection methods often require expensive instruments or require radiolabelling or enzymatic labelling of the catalytic substrate. It is well known that some animal experiments are very time consuming and expensive and may even be dangerous to the health of the animal's life. The electrochemical method has the advantages of simple equipment, low price, high sensitivity, good specificity, effectiveness and the like.
Disclosure of Invention
The invention aims to combine the nucleic acid modified gold nanoparticles, exert the advantages of an electrochemical detection technology and establish an effective and extremely high-sensitivity electrochemical detection method for the avian infectious bronchitis virus H120 strain.
The technical scheme of the invention is as follows: the invention relates to an electrochemical detection method of chicken infectious bronchitis virus H120 strain. The method comprises the steps of equivalently converting RNA of an infectious bronchitis virus H120 strain into DNA (deoxyribonucleic acid) capable of specifically identifying the RNA of the H120 strain, namely H120-target, by annealing, enzyme digestion with S1nuclease (S1) and high-temperature heating, fixing a sulfydryl modified nucleic acid probe 2 on the surface of a gold electrode to incubate with the H120-target in a self-assembly mode, introducing a nucleic acid probe 1 modified gold nanoparticle capable of being specifically combined with the H120-target to adsorb an electric signal molecule ruthenium hexamine (RuHex) for generation and signal amplification, finally carrying out electrochemical detection on the adsorbed RuHex by using a linear voltammetry method, obtaining a linear equation by drawing a standard relation curve between the concentration of the H120 strain and an electric signal, and calculating the content of the H120 strain in an actual sample according to the size of the detected electric signal.
The method comprises the following steps: the method comprises the following steps of AuNPs preparation, probe 1 modified AuNPs preparation, gold electrode pretreatment, probe 2 modified gold electrode preparation, gold electrode and sample hybridization, gold electrode and AuNPs co-incubation and electrochemical detection.
(1) Preparation of AuNPs
All glassware had to be newly treated with aqua regia (HNO) before preparation3Soaking in HCl 3: 1) for 30min, washing with distilled water, and oven drying. The preparation process of AuNPs is as follows: 100mL of a 0.01% chloroauric acid solution was added to a three-necked flask and heated to boiling with stirring. And after the solution is boiled, rapidly adding 3.5mL of 1% trisodium citrate, continuously heating and stirring for 15min, turning off the heater after the solution turns to wine red, continuously stirring for 30min, turning off the stirrer, cooling the solution to room temperature, and storing the prepared AuNPs in a brown reagent bottle at 4 ℃.
(2) Preparation of Probe 1 modified AuNPs
mu.L of AuNPs prepared in (1) was put in a 2.0mL round-bottom centrifuge tube, 40. mu.L of 10. mu.M probe 1 was added thereto, and 360. mu.L of synthetic buffer (10mM PBS, 0.01M NaCl, pH 7.0) was added thereto, and the solution was mixed well and incubated at 37 ℃ for 12 hours. 12000r/min, centrifuging for 30min at 4 ℃, then discarding the supernatant, resuspending by using a synthetic buffer, repeating for 3 times to obtain 500 mu L of AuNPs with the surface modified by the probe 1, and storing for later use at 4 ℃.
The sequence of the probe 1 is as follows: 5 '-SH-TTT TTT TCA GGT GAG TTA-3'.
(3) Pretreatment of gold electrodes
Using goldfish (concentrated sulfuric acid: H) to make gold electrode use2O2In a volume ratio of 3: 1) and 50% nitric acid solution for 5min and 30 min. Polishing the electrodes with 2000 mesh and 5000 mesh sandpaper for 5min, polishing the gold electrodes with 1 μm and 0.3 μm aluminum oxide powder, and performing ultrasonic treatment with alcohol and ultrapure water for 5 min. The treated electrode was placed at 0.5M H2SO4In the voltage range of 0V-1.5V, cyclic voltammetry scanning is carried out for 30 circles, and the scanning speed parameter is set to be 0.1V/s.
(4) Preparation of Probe 2 modified electrode
mu.L of 10. mu.M probe 2 and 180. mu.L of immobilized Buffer (10mM Tris-HCl, 1mM EDTA, 10mM TCEP, 0.1M NaCl, pH 7.4) were mixed well in a centrifuge tube. 50 μ L of the above solution was put into a new 2.0mL round-bottom centrifuge tube, dipped in the gold electrode solution of (3) above, and incubated for 12 h. The electrode was rinsed with double distilled water, and then rinsed after soaking the electrode in 1mM mercaptohexanol solution for 1 h.
The sequence of the probe 2 is as follows: 5 '-GAT CAT AAT ATA TAT ATA T-SH-3'.
(5) Hybridization of gold electrodes with samples
The virus liquid of 200 mu L H120 strain was used to extract viral RNA to obtain 40 mu L of 1.56e-6mu.M of H120 strain RNA. Diluting to obtain 1.56e-7μM、1.56e-8μM、1.56e-9μ M of standard solution. mu.L of viral RNA with different concentrations, 2. mu.L of 10. mu. M H120-target and 85. mu.L of hybrid Buffer (10mM PBS, 0.25M NaCl, pH 7.0) were mixed well, subjected to water bath at 90 ℃ for 5min, slowly annealed, and naturally cooled to room temperature, so that H120-target and H120-RNA were hybridized. Subsequently, 1. mu. L S1 nucleic (80U/. mu.L), 10. mu.L of 10 XS 1 nucleic Buffer were added to each sample tube and mixed, and reacted at 37 ℃ for 30min, after 90 ℃ for 15min, the gold electrode in (4) above was immersed in the mixed system and annealed for hybridization.
The sequence of the H120-target is as follows: 5'-ATT ATG ATC TAA CTC ACC TGA-3' are provided.
(6) Gold electrodes incubated with AuNPs
And (3) cleaning the gold electrode in the step (5) by using double distilled water, placing the cleaned gold electrode in 50 mu L of the AuNPs solution which is prepared in the step (2) and is provided with the probe 1 on the surface, and carrying out water bath at 30 ℃ for 2 h.
(7) Electrochemical detection
The electrode treated in (6) above was washed with double distilled water, and the electrode was placed in a solution of 10mM Tris-HCl, 50. mu.M RuHex (pH 7.0) for quantitative determination by electroanalysis. The detection adopts an electrochemical workstation CHI 660E and a three-electrode system consisting of a saturated silver chloride reference electrode, a platinum counter electrode and a gold electrode. The scanning method used was Linear Sweep Voltammetry (LSV), with a potential set to-0.65V to 0.1V and a sweep rate set to 0.5V/s. According to the logarithm of the concentration of H120 strain and LSV500The mV current value (ip) is used for making a standard curve, and the magnitude of an electric signal is detected to calculate to obtain the current value (ip) to be measured in the actual sampleAnd (5) measuring the content of the substance.
H120 strain virus liquid is 1.56e-9μM-1.56e-6In the range of μ M concentration (minimum virus detection limit of 1.56 e)-9Mu M), the logarithm of the virus liquid concentration and the current value (ip) have good linear relation, and the quantitative requirement is met.
The invention has the beneficial effects that: the method designs and utilizes the characteristic nucleotide H120-target for specifically recognizing the RNA of the H120 strain, converts the H120-target and the RNA of the chicken infectious bronchitis virus H120 strain in an equivalent manner by means of S1 nucleic enzyme digestion and high-temperature heating at 90 ℃, avoids the degradation of the RNA in the subsequent detection process, simultaneously utilizes a self-assembly manner to modify a probe 2 on the surface of a gold electrode, takes gold nanoparticles modified by the probe 1 as signal amplification, combines the advantages of sensitivity and convenience of an electrochemical detection method, realizes the sensitive detection of the H120 strain of the chicken infectious bronchitis virus, and has important significance for the sensitive detection of the H120 strain of IBV.
Drawings
FIG. 1 is a schematic diagram of the electrochemical detection method of IBV H120 strain
FIG. 2 is a feasibility verification diagram of the electrochemical detection method for IBV H120 strain
FIG. 3 is a graph of the logarithmic relationship between the electrochemical signal and the concentration of H120 strain
Detailed Description
Example 1 feasibility verification of electrochemical detection method for IBV H120 strain
Under the best experimental conditions, system specificity verification is carried out. And (4) replacing the IBV H120 strain RNA stock solution in the step (5) with IBV NNA strain RNA stock solution and double distilled water without RNase to verify the feasibility of the detection system according to the method. Specifically, the RNA stock solution of IBV H120 strain, the RNA stock solution of IBVNNA strain and RNase Free ddH are taken2To 2. mu.L of each O, 2. mu.L of 10. mu. M H120-target and 85. mu.L of hybridization Buffer (10mM PBS, 0.25M NaCl, pH 7.0) were added and mixed, and the experiment was carried out according to the above-mentioned experimental procedures (1) to (7). Electrochemical scanning was performed under optimal experimental conditions. The results are shown in fig. 2A-B, and the detection system detects H120 strains, but cannot detect NNA strains, thus confirming that the detection system has feasibility, good specificity and no cross reaction.
Example 2 determination of electrochemical Signal-logarithmic concentration Standard Curve for different concentrations of Virus fluid
The preparation concentrations in the step (5) of the method are respectively 1.56e-9μM、1.56e-8μM、1.56e-7μM、1.56e-6mu.M H120 virus sample, and the prepared sample is subjected to linear voltammetry scanning under the optimal experimental conditions. As a result, as shown in FIG. 3A, the peak current value increased as the virus concentration increased. The virus liquid is 1.56e-9μM-1.56e-6Log of virus concentration and LSV in the μ M range500The mV current values (ip) are well linear (fig. 3B), both of which correspond to y being 7.9821+0.6406lgx (R)20.99837), wherein y is electrochemical signal peak (muA), x is H120 virus concentration (muM), and the method can be used as the basis for quantitative detection of IBV H120 in actual samples.
Claims (6)
1. The electrochemical detection method of the chicken infectious bronchitis virus H120 strain is characterized by comprising three probes which take RNA of the chicken infectious bronchitis virus H120 strain as a target: probe 1, probe 2, H120-target;
wherein:
1, probe 1: 5 '-SH-TTT TTT TCA GGT GAG TTA-3'
And (3) probe 2: 5 '-GAT CAT AAT ATA TAT ATA T-SH-3'
H120-target：5’-ATT ATG ATC TAA CTC ACC TGA-3’
The method comprises the construction of an electrochemical sensor, and comprises the following steps: incubating a nucleic acid probe 2 fixed on the surface of a gold electrode in a self-assembly manner with H120-target to obtain a probe 2/H120-target modified gold electrode, introducing gold nanoparticles of a modified nucleic acid probe 1 specifically combined with H120-target to adsorb an electric signal molecule ruthenium hexamine RuHex for generation and signal amplification, finally performing electrochemical detection on the adsorbed RuHex by using a linear voltammetry method, and drawing a standard relation curve between H120 strain concentration and an electric signal to obtain a linear equation so as to complete the construction of an electrochemical sensor;
the detection method is that the content of the H120 strain in the actual sample is calculated according to the detected current intensity.
2. The electrochemical detection method of the chicken infectious bronchitis virus H120 strain according to claim 1, characterized in that RNA of the chicken infectious bronchitis virus H120 strain is equivalently converted into DNA capable of specifically recognizing the RNA of the H120 strain, namely H120-target, by annealing, S1nuclease S1 enzyme digestion and high-temperature heating, 2 μ L of 10 μ M H120-target, 2 μ L of extracted H120 strain RNA, 10mM PBS and 0.25M NaCl in 85 μ L DEPC water and a hybridization solution with the pH value of 7.0 are mixed, the mixture is slowly cooled to room temperature in 90 ℃ water bath for 5min and annealed to form a hybrid double strand, then 1 μ L S1 and 10 μ L of 10 × S1 enzyme digestion solution are added into the solution to carry out enzyme digestion at 37 ℃ for 30min, and after the high temperature of 90 ℃ for 15min, the virus RNA is equivalently converted into H120-target.
3. The electrochemical detection method of the chicken infectious bronchitis virus H120 strain according to claim 1, characterized in that the probe 2/H120-target modified gold electrode is prepared by placing the pretreated gold electrode in 50 μ L of a 0.2 μ M probe 2, 10mM Tris-HCl, 1mM EDTA, 0.01M NaCl, pH 7.0 fixing solution, incubating at room temperature for 12H, washing to clean, soaking the electrode in 100 μ L of a 1mM mercaptohexanol solution, incubating at room temperature in the dark for 1H, washing the electrode, soaking the electrode in the H120-target solution of claim 2, and hybridizing for 2H.
4. The electrochemical detection method of the chicken infectious bronchitis virus H120 strain according to claim 1, characterized in that, gold nanoparticles of a modified nucleic acid probe 1 capable of specifically binding with H120-target are introduced to adsorb an electric signal molecule hexamine RuHex for generation and signal amplification, in an experiment, 100 μ L of gold nanoparticles, 40 μ L of 10 μ M of nucleic acid probe 1 are mixed with 360 μ L of synthetic solution containing 10mM PBS, 0.01M NaCl and pH 7.0, after incubation at 37 ℃ for 12H, centrifugation is performed at 4 ℃ 12000r/min for 30min, the synthetic solution is washed and resuspended three times to obtain 500 μ L of gold nanoparticles of a surface modified probe 1, and 50 μ L of gold nanoparticles of the modified nucleic acid probe 1 are taken to incubate with the probe 2/H120-target modified gold electrode of claim 3 at 30 ℃ for 2H.
5. The method for electrochemically detecting the infectious bronchitis virus H120 strain of chicken as claimed in claim 1, wherein the electrochemical detection of the adsorbed RuHex is carried out by linear voltammetry, a gold electrode modified by the probe 2/H120-target/probe 1 in claim 4 is cleaned and then placed in 5mL of a buffer solution containing 10mM Tris-HCl and 50 μ M RuHex and having a pH value of 7.0 for electrochemical quantitative detection, the potential is set to-0.65V to 0.1V, and the sweeping speed is 500 mV.
6. The electrochemical detection method of the H120 strain of the avian infectious bronchitis virus of claim 1, wherein a linear equation is obtained by plotting a standard relation curve between the concentration of the H120 strain and an electric signal, wherein y is 7.9821+0.6406lgx, R is20.99837, wherein y is electrochemical signal peak value muA, x is H120 strain virus liquid concentration muM, and H120 strain content in the actual sample is calculated according to the detected electric signal.
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