CN110779970B - A kind of electrochemical detection method of chicken infectious bronchitis virus H120 strain - Google Patents

Abstract

The invention belongs to the technical field of analytical chemistry, and relates to an electrochemical detection method and application of a chicken infectious bronchitis virus H120 strain. The present invention converts the RNA of chicken infectious bronchitis virus H120 strain into DNA that can specifically recognize H120 strain RNA, namely H120-target, by means of annealing, S1 nuclease (S1) enzymatic cleavage and high-temperature heating, and is based on self-assembly. The sulfhydryl-modified nucleic acid probe 2 is immobilized on the surface of the gold electrode and incubated with H120-target, and the gold nanoparticles modified by the nucleic acid probe 1 that can specifically bind to the H120-target are introduced to adsorb the electrical signal molecule RuHexammine (RuHex ) is used to generate and amplify the signal, and finally the adsorbed RuHex is electrochemically detected by linear voltammetry, the linear equation is obtained by drawing the standard relationship curve between the concentration of H120 strain and the electrical signal, and the actual electrical signal is calculated according to the magnitude of the detected electrical signal. The content of the H120 strain in the sample. This method has the advantages of low cost, high sensitivity and strong specificity.

Description

A kind of electrochemical detection method of chicken infectious bronchitis virus H120 strain

technical field

The invention belongs to the technical field of analytical chemistry, and relates to an electrochemical method for detecting chicken infectious bronchitis virus H120 strain.

Background technique

Chicken infectious bronchitis is an acute, highly contagious infectious disease caused by the coronavirus genus Infectious Bronchitis Virus (IBV), which causes huge economic losses to the chicken industry worldwide. IBV is a single-stranded virus whose genes can be mutated by point mutation and recombination. There are many IBV serotypes, and there is no or little cross-reaction between different serotypes, which increases the difficulty of immune prevention. Therefore, it is of great practical significance to establish an effective method to detect different strains of IBV to assist in the selection of suitable vaccines.

Nanotechnology has become a hot spot in the field of materials research today. Gold nanoparticles are one of the most stable nanoparticles among metal nanoparticles, which have good stability, small size effect, surface effect, optical effect and good biocompatibility, etc. Features. Based on the self-assembly of gold and sulfhydryl groups, gold nanoparticles can form probes with specific DNA sequences modified with sulfhydryl groups, or DNA-modified electrodes can be prepared by self-assembly of sulfhydryl groups on the surface of gold, which are important in the fields of biomedicine, gene therapy, and clinical testing. effect.

Nowadays, the commonly used methods for virus detection include immunoprecipitation, fluorescence quantitative PCR, and enzyme-linked immunosorbent assay. These detection methods often require expensive equipment or require radiolabeling or enzymatic labeling of catalytic substrates. As we all know, some animal experiments are very time-consuming and expensive, and may even endanger the life and health of animals. Electrochemical methods have the advantages of simple equipment, low price, high sensitivity, good specificity, and effectiveness.

SUMMARY OF THE INVENTION

The purpose of the present invention is to combine nucleic acid-modified gold nanoparticles, exert the advantages of electrochemical detection technology, and establish an effective and extremely sensitive electrochemical detection method for chicken infectious bronchitis virus H120 strain.

Technical scheme of the present invention: The present invention is an electrochemical detection method for the H120 strain of infectious bronchitis virus of chickens. The present invention converts the RNA of chicken infectious bronchitis virus H120 strain into DNA that can specifically recognize H120 strain RNA, namely H120-target, by means of annealing, S1 nuclease (S1) enzyme cleavage and high-temperature heating, and is based on self-assembly. The sulfhydryl-modified nucleic acid probe 2 was immobilized on the surface of the gold electrode and incubated with H120-target, and the gold nanoparticles modified with the nucleic acid probe 1 that could specifically bind to the H120-target were introduced to adsorb the electrical signal molecule RuHexammine (RuHex ) is used to generate and amplify the signal, and finally the adsorbed RuHex is electrochemically detected by linear voltammetry, the linear equation is obtained by drawing the standard relationship curve between the concentration of H120 strain and the electrical signal, and the actual electrical signal is calculated according to the magnitude of the detected electrical signal. The content of the H120 strain in the sample.

The method includes the following steps: preparation of AuNPs, preparation of AuNPs modified by probe 1, pretreatment of gold electrode, preparation of gold electrode modified by probe 2, hybridization of gold electrode and sample, co-incubation of gold electrode and AuNPs, and electrochemical detection.

(1) Preparation of AuNPs

Before preparation, all glassware must be soaked in newly configured aqua regia ( _HNO3 :HCl=3:1) for 30min, rinsed with a large amount of double-distilled water, and dried for use. The preparation process of AuNPs was as follows: 100 mL of 0.01% chloroauric acid solution was added into a three-necked flask and heated to boiling with stirring. After it boils, quickly add 3.5mL of 1% trisodium citrate, continue to heat and stir for 15min, turn off the heater when the solution turns wine red, continue to stir for 30min, turn off the stirrer, and the solution will be prepared after cooling to room temperature The AuNPs were stored in a brown reagent bottle at 4°C.

(2) Preparation of probe 1 modified AuNPs

Take 100 μL of the AuNPs prepared in (1) into a 2.0 mL round-bottomed centrifuge tube, add 40 μL of 10 μM probe 1, and then add 360 μL of synthetic buffer (10 mM PBS, 0.01 M NaCl, pH=7.0), and mix the solution evenly Incubate at 37°C for 12h. After centrifugation at 12000 r/min for 30 min at 4°C, the supernatant was discarded, resuspended in synthetic buffer, and repeated 3 times to obtain 500 μL of AuNPs with surface-modified probe 1, which was stored at 4°C for later use.

The sequence of the above probe 1 is: 5'-SH-TTT TTT TCA GGT GAG TTA-3'.

(3) Pretreatment of gold electrodes

The gold electrodes were treated with piranha ( ₃ : ₁ volume ratio of concentrated sulfuric acid:H2O2) and 50% nitric acid solution for 5 min and 30 min, respectively. After grinding the electrodes with 2000-mesh and 5000-mesh sandpaper for 5 min, respectively, the gold electrodes were polished with 1 μm and 0.3 μm aluminum oxide powder, and ultrasonicated with alcohol and ultrapure water for 5 min respectively. The treated electrodes were placed in 0.5MH ₂ SO ₄ , and cyclic voltammetry scanning was performed for 30 cycles in the voltage range of 0V-1.5V, and the scanning speed parameter was set to 0.1V/s.

(4) Preparation of probe 2 modified electrode

Take 20 μL of 10 μM probe 2 and 180 μL of immobilization buffer (10 mM Tris-HCl, 1 mM EDTA, 10 mM TCEP, 0.1 M NaCl, pH=7.4) in a centrifuge tube and mix well. Take 50 μL of the above solution into a new 2.0 mL round-bottomed centrifuge tube, immerse it in the gold electrode solution in (3) above, and incubate for 12 h. Rinse with double distilled water, then soak the electrode in 1 mM mercaptohexanol solution for 1 h and rinse.

The sequence of the above probe 2 is: 5'-GAT CAT AAT ATA TAT ATA T-SH-3'.

(5) Hybridization between gold electrode and sample

Take 200 μL of H120 strain virus solution for extraction of viral RNA, and obtain 40 μL of H120 strain RNA with a concentration of 1.56e- ⁶ μM. It was diluted to obtain standard solutions with concentrations of 1.56e ^-7 μM, 1.56e- ⁸ μM, and 1.56e ^-9 μM. Take 2 μL of different concentrations of viral RNA, 2 μL of 10 μM H120-target and 85 μL of hybridization buffer (10 mM PBS, 0.25 M NaCl, pH=7.0), mix well, bathe at 90 °C for 5 min, slowly anneal and naturally cool to room temperature, so that H120-target and H120-RNA hybridization. Immediately add 1 μL of S1 Nuclease (80U/μL) and 10 μL of 10×S1 Nuclease Buffer to each sample tube to mix well, react at 37°C for 30min and 90°C for 15min, then immerse the gold electrode in (4) above into the mixture. System annealing hybridization.

The sequence of the above H120-target is: 5'-ATT ATG ATC TAA CTC ACC TGA-3'.

(6) Incubation of gold electrodes with AuNPs

The gold electrode in the above (5) was washed with double distilled water, and placed in 50 μL of the AuNPs solution with the surface-modified probe 1 prepared in the above (2), in a water bath at 30 °C for 2 h.

(7) Electrochemical detection

The electrode treated in (6) above was rinsed with double distilled water, and the electrode was placed in a solution of 10 mM Tris-HCl, 50 μM RuHex (pH=7.0) for quantitative detection by electroanalysis. The detection adopts electrochemical workstation CHI 660E, a three-electrode system composed of saturated silver chloride reference electrode, platinum counter electrode and gold electrode. The sweep method used was linear sweep voltammetry (LSV), the potential was set from -0.65V to 0.1V, and the sweep speed was set at 0.5V/s. According to the logarithm of the H120 strain concentration and the current value (ip) of LSV ₅₀₀ mV, a standard curve was made, and the content of the substance to be tested in the actual sample could be calculated by detecting the magnitude of the electrical signal.

The virus solution of H120 strain is in the concentration range of 1.56e ^-9 μM-1.56e ^-6 μM (the minimum detection limit of virus solution is 1.56e ^-9 μM), and the logarithm of the virus solution concentration and the current value (ip) have a good linearity relationship, meet quantitative requirements.

Beneficial effects of the present invention: The method designs and utilizes specificity to identify the characteristic nucleotide H120-target of H120 strain RNA, and separates H120-target and H120 strain of chicken-borne branch virus by S1 Nuclease digestion and high temperature heating at 90°C. The RNA is converted into an equal amount to avoid the degradation of RNA in the subsequent detection process. At the same time, probe 2 is modified on the surface of the gold electrode by self-assembly, and the gold nanoparticles modified by probe 1 are used as signal amplification. Combined with the sensitive electrochemical detection method , The advantage of convenience, the sensitive detection of H120 strain of chicken infectious bronchitis virus is realized, which is of great significance to the sensitive detection of H120 strain of IBV.

Description of drawings

Figure 1 is a schematic diagram of the electrochemical detection method of IBV H120 strain

Figure 2 shows the feasibility verification diagram of the electrochemical detection method for IBV H120 strain

Fig. 3 is the logarithmic relationship between electrochemical signal and H120 strain concentration

Detailed ways

Example 1. Feasibility verification of electrochemical detection method of IBV H120 strain

The system specificity was verified under the best experimental conditions. According to the above method, the IBV H120 strain RNA stock solution in step (5) was replaced by the IBV NNA strain RNA stock solution and double distilled water without RNase to verify the feasibility of the detection system. Specifically, take 2 μL each of IBV H120 strain RNA stock solution, IBVNNA strain RNA stock solution and RNase Free ddH ₂ O, add 2 μL 10 μM H120-target and 85 μL hybridization buffer (10 mM PBS, 0.25 M NaCl, pH=7.0), respectively, and mix well , according to the above experimental steps (1) to (7) to carry out the experiment. Gauge electrochemical scans were performed under optimal experimental conditions. The results are shown in Figure 2A-B, the detection system detected the H120 strain, but could not detect the NNA strain, which confirmed that the detection system was feasible, specific, and had no cross-reaction.

Embodiment 2. Determination of the electrochemical signal-concentration logarithmic standard curve of the virus liquid of different concentrations

The H120 virus samples with concentrations of 1.56e ^-9 μM, 1.56e- ⁸ μM, 1.56e ^-7 μM, and 1.56e- ⁶ μM were prepared in step (5) of the above method, and the prepared samples were placed in the best experiment. A linear voltammetry scan was performed under the conditions. The results are shown in Fig. 3A, when the virus concentration increased, the peak current value increased. In the range of 1.56e ^-9 μM-1.56e ^-6 μM, the logarithm of the virus concentration and the current value (ip) of LSV ₅₀₀ mV have a good linear relationship (Figure 3B), and the two are in line with y=7.9821+0.6406 lgx (R ² =0.99837), where y is the peak value of electrochemical signal (μA), and x is the concentration of H120 strain virus solution (μM), which can be used as the basis for quantitative detection of IBV H120 strain in actual samples.

Claims (6)

1. the electrochemical detection method of a chicken infectious bronchitis virus H120 strain, is characterized in that, this detection method comprises three probes that are target for the RNA of chicken infectious bronchitis virus H120 strain: probe 1, probe Needle 2, H120-target; in: Probe 1: 5'-SH-TTT TTT TCA GGT GAG TTA-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 includes the construction of an electrochemical sensor, and the steps are as follows: the nucleic acid probe 2 fixed on the surface of the gold electrode by self-assembly is incubated with H120-target to obtain a gold electrode modified by probe 2/H120-target, and then introduced into the gold electrode modified with H120-target. The gold nanoparticles of modified nucleic acid probe 1 specifically bound to the target are used to adsorb the electrical signal molecule RuHex, which is used to generate and amplify the signal. Finally, the adsorbed RuHex is electrochemically detected by linear voltammetry. By drawing the H120 strain A linear equation is obtained from the standard relationship curve between the concentration and the electrical signal, and then the construction of the electrochemical sensor is completed; The detection method is as follows: calculating the content of the H120 strain in the actual sample according to the magnitude of the current intensity obtained by the detection. 2. the electrochemical detection method of a kind of chicken infectious bronchitis virus H120 strain according to claim 1, is characterized in that, by annealing, S1 nuclease S1 enzymatic cutting and high temperature heating mode by chicken infectious bronchitis virus H120 The RNA of the strain was converted into H120-target, the DNA that can specifically recognize the RNA of the H120 strain, and 2 μL of 10 μM H120-target, 2 μL of the extracted H120 strain RNA and 85 μL of DEPC water were prepared with 10 mM PBS and 0.25 M NaCl, Hybridization solution with pH value of 7.0 was mixed, cooled in a water bath at 90°C for 5 min to form a hybrid double-stranded annealing at room temperature, and then 1 μL of S1 and 10 μL of 10×S1 enzyme digestion solution were added to the above solution, digested at 37°C for 30 minutes, and high temperature at 90°C for 15 minutes After that, the equal amount of viral RNA can be converted into H120-target. 3. the electrochemical detection method of a kind of chicken infectious bronchitis virus H120 strain according to claim 1, is characterized in that, the preparation method of probe 2/H120-target modified gold electrode, the gold electrode of pretreatment is placed Incubate for 12 h at room temperature in 50 μL of 0.2 μM probe 2, 10 mM Tris-HCl, 1 mM EDTA, 0.01 M NaCl, pH 7.0 in a fixative solution at room temperature, rinse it off, and then soak the electrode in 100 μL of 1 mM mercaptohexanol solution at room temperature to protect from light After 1 h of incubation, the electrode was cleaned, and it was immersed in the H120-target solution of claim 2 for hybridization for 2 h. 4. the electrochemical detection method of a kind of chicken infectious bronchitis virus H120 strain according to claim 1 is characterized in that, the gold nanoparticle of the modified nucleic acid probe 1 that can be specifically combined with H120-target is introduced to absorb electricity. The signal molecule hexaammine RuHex was used to generate and amplify the signal. In the experiment, 100 μL gold nanoparticles, 40 μL 10 μM nucleic acid probe 1 and 360 μL synthesis solution containing 10 mM PBS, 0.01 M NaCl, pH 7.0 were mixed at 37°C. After 12 hours of incubation, centrifuge at 12000 r/min at 4°C for 30 minutes, wash and resuspend the synthetic solution three times to obtain 500 μL of gold nanoparticles of surface-modified probe 1, and take 50 μL of gold nanoparticles of modified nucleic acid probe 1 and probe 2 of claim 3 /H120-target modified gold electrodes were incubated at 30°C for 2h. 5. the electrochemical detection method of a kind of chicken infectious bronchitis virus H120 strain according to claim 1, it is characterized in that utilizing linear voltammetry to carry out electrochemical detection to the RuHex of adsorption, in claim 4, probe 2 After cleaning the gold electrode modified by /H120-target/probe 1, it was placed in 5mL buffer solution containing 10mM Tris-HCl, 50μM RuHex, pH 7.0 for electrochemical quantitative detection, and the potential was set from -0.65V to 0.1 V, sweep speed 500mV. 6. the electrochemical detection method of a kind of chicken infectious bronchitis virus H120 strain according to claim 1, is characterized in that by drawing the standard relation curve between H120 strain concentration and electrical signal to obtain linear equation, y=7.9821+ 0.6406lgx, R ² =0.99837, where y is the peak value of the electrochemical signal μA, x is the concentration μM of the H120 strain virus solution, and the content of the H120 strain in the actual sample is calculated according to the electrical signal obtained from the detection.

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