Multi-recurrent tick-borne virus detection chip as well as preparation method and detection method thereof

Abstract

The invention is applicable to the technical field of virus detection, and provides a multi-recurrent tick-borne virus detection chip and a preparation method and a detection method.The multi-recurrent tick-borne virus detection chip comprises a substrate, gold nanoparticles are deposited on the substrate to form a fluorescence enhancement platform, and six kinds of sulfydryl-modified single-stranded nucleic acid probes are loaded on the fluorescence enhancement platform and are used for detecting tick-borne viruses TBEV, ALSV, SFTSV, MKWV, BJNV and YEZV respectively. The sequence of the gene is as shown in SEQ NO.1-6, and the 5'end is modified with sulfydryl and 12 adenine bases. According to the invention, amplified cDNA reaches single copy sensitivity, single-chain amplification products of six tick-borne viruses can be specifically detected, and cross reaction is avoided; by taking qPCR as a gold standard, the chip accurately recognizes 11 positive tick samples and 66 negative tick samples, and shows 91.6% of sensitivity and 100% of specificity in tick-borne virus nucleic acid detection.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, and in particular relates to a detection chip for multiple re-emerging tick-borne viruses, as well as its preparation method and detection method. Background Technology

[0002] Tick-borne viruses are a class of insect-borne viruses that seriously endanger human and animal health. Currently, in addition to common tick-borne viruses such as tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, and fever with thrombocytopenia syndrome virus, new viruses such as Alongshan virus, Songling virus, and Arctic Nairo virus have been discovered. Newly emerging tick-borne viruses, such as Ji'an Nairo virus, Yichun Nairo virus, and Yichun sandfly virus, have the potential to infect humans and are a new and unknown safety threat factor, as well as an important public health issue.

[0003] In the detection of emerging tick-borne viruses, accurate nucleic acid testing is the gold standard for viral diagnosis and is crucial for the diagnosis, prognosis, and basic research of emerging infectious diseases. Traditional molecular detection methods, such as multiplex PCR and loop-mediated isothermal amplification (LAM), can achieve simultaneous detection of multiple pathogens, meeting basic needs. However, they have an upper limit to the types of pathogens they can detect, and their sensitivity and specificity decrease significantly as the number of detectable targets increases. Although high-throughput sequencing has the ability to detect multiple pathogens, its high cost, long testing cycle, and relatively low sensitivity limit its clinical application. Therefore, there is an urgent need for a detection technology that is simple to operate, rapid, efficient, highly sensitive, and has high throughput to achieve simultaneous screening of multiple tick-borne viruses.

[0004] Compared with currently used nucleic acid analysis technologies such as quantitative real-time PCR and high-throughput sequencing, plasma-enhanced fluorescence chips can perform multiplex nucleic acid molecular detection using probes labeled with different substrates, and have advantages such as specificity, high throughput and low reagent consumption. Summary of the Invention

[0005] The purpose of this invention is to provide a multi-recurrent tick-borne virus detection chip, which aims to solve the problems mentioned in the background art.

[0006] The present invention is implemented as follows: a multi-recurrent tick-borne virus detection chip includes a substrate on which gold nanoparticles are deposited to form a fluorescence enhancement platform. The fluorescence enhancement platform is loaded with six thiol-modified single-stranded nucleic acid probes, which are respectively targeted at tick-borne viruses TBEV, ALSV, SFTSV, MKWV, BJNV, and YEZV, and their sequences are shown in SEQ NO.1-6. The probes are modified with a thiol group and 12 adenine bases at the 5' end.

[0007] Another objective of this invention is to provide a method for preparing a detection chip for multiple recurrent tick-borne viruses, comprising the following steps: The glass substrate was pretreated by immersing it in a mixed solution of concentrated H2SO4 and 30% H2O2 for cleaning. Add 1% HAuCl4·3H2O to deionized water, heat to boiling and stir vigorously, then quickly add 2% sodium citrate solution, continue the reaction and cool to room temperature to obtain a gold nanoparticle colloidal solution. The pretreated glass substrate was immersed in a gold nanoparticle ion colloidal solution. Hexane was introduced to form an oil-water interface. Ethanol was injected to promote the self-assembly of gold nanoparticles at the interface to form a dense monolayer. The colloidal suspension was discharged from the bottom, allowing the nanoparticle monolayer on the interface to transfer to the glass substrate. The modified substrate was then heat-treated under argon protection to obtain a fluorescence enhancement platform. Six thiol-modified single-stranded nucleic acid probes were immobilized on a fluorescence enhancement platform. The probes were respectively targeted at tick-borne viruses TBEV, ALSV, SFTSV, MKWV, BJNV, and YEZV, and their sequences are shown in SEQ NO.1-6. The probes were modified with thiol groups and 12 adenine bases at the 5' end.

[0008] Another objective of this invention is to provide a detection method for multiple recurrent tick-borne virus detection chips, comprising the following steps: The RNA of the test sample was reverse transcribed into cDNA, and PCR amplification was performed using a 5'-biotin-labeled forward primer and a 5'-phosphorylated reverse primer. The amplification product was digested with λ exonuclease, which specifically cleaves phosphorylated double-stranded DNA to produce biotinylated single-stranded amplification products. The biotinylated single-stranded amplification product was incubated on a fluorescence enhancement platform and captured by an immobilized single-stranded nucleic acid probe. It was then detected and identified using a streptavidin-conjugated near-infrared fluorescent dye SA-IRDye800CW, and the signal value was detected using a scanner.

[0009] The multi-recurrent tick-borne virus detection chip provided in this invention is a plasma-enhanced fluorescence (PEF) platform functionalized with six detection probes. Specific labeling is achieved using IRDye800CW fluorescent dye. This PEF platform achieves a 50-fold increase in fluorescence signal and exhibits a wide linear detection range spanning five orders of magnitude, with a limit of detection (LOD) ranging from 5.62 to 9.91 pM. The detection method provided in this invention achieves single-copy sensitivity for amplified cDNA and can specifically detect single-stranded amplification products of six tick-borne viruses without cross-reactivity. Using qPCR as the gold standard, the chip accurately identified 11 positive tick samples and 66 negative tick samples, demonstrating a sensitivity of 91.6% and a specificity of 100% in tick-borne virus nucleic acid detection. Attached Figure Description

[0010] Figure 1 This is a flowchart illustrating the preparation process of the fluorescence enhancement platform provided in Example 1 of the present invention; Figure 2 , Figure 3 The results of ssDNA detection using the fluorescence enhancement platform provided in Embodiment 1 of the present invention are shown below. Figure 2 In the diagram, 'a' represents the dilution procedure for standard single-stranded DNA. Figure 2 Image b shows IRDye800 fluorescence images of ALSV-targeting single-stranded DNA detected on a fluorescence enhancement platform and a glass substrate. Figure 3 c is based on Figure 2 The curve of IRDye800 fluorescence intensity versus target single-stranded DNA concentration is plotted in b. Figure 3 In the figure, d represents the slope of the calibration curve for the detection of virus-specific single-stranded DNA of TBEV, SFTSV, MKWV, BJNV, and YEZV on the fluorescence enhancement platform. Figure 4 The single-stranded DNA of the PCR product and the corresponding probe sequence in the tick-borne virus combination provided in Example 2 of this invention; Figure 5 The results of detection of different single-stranded amplicon provided in Example 2 of the present invention are shown in Figure b, which is the IRDye800 fluorescence imaging image of tick-borne virus single-stranded DNA in PCR products identified by the fluorescence enhancement platform; Figure c is the identification heatmap of different tick-borne virus single-stranded DNA samples; and Figure d is the sensitivity result of gradient dilution detection of BJNV virus by fluorescence enhancement detection method. Figure 6 The heatmap shows 11 positive samples and 67 negative samples provided in Embodiment 3 of the present invention. Figure 7 This is a scatter plot of viral single-stranded DNA signal levels in the positive group (red) and negative group (black) provided in Example 3 of the present invention, where a is TBEV; b is ALSV; c is SFTSV; d is MKWV; e is BJNV; and f is YEZV virus. Figure 8 The results of the detection of tick samples provided in Example 3 of the present invention are as follows: a) is the fluorescence intensity of the positive sample measured by the fluorescence enhancement detection method; b) is the consistency comparison between the fluorescence enhancement detection method and the sensitivity of qPCR, and a Ct value > 30 in qPCR detection is considered negative; c) is the correlation analysis results between the detected fluorescence signal and the virus titer calculated by qPCR. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0012] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0013] Example 1: A fluorescence enhancement platform, such as Figure 1 As shown, its preparation method includes the following steps: For glass substrates (2.5 cm × 7.5 cm) 2 First, perform interface pretreatment by placing it in a mixed solution of concentrated H2SO4 and 30% H2O2 (volume ratio 7:3) and immersing and washing it at 140℃ for 1 hour. The preparation of gold nanoparticles was carried out according to existing methods with slight adjustments: 2 mL of 1% HAuCl4·3H2O was added to 198 mL of deionized water, heated to boiling and stirred vigorously, and then 1 mL of 2% sodium citrate solution was quickly injected. After reacting for 20 minutes, the mixture was cooled to room temperature to obtain a colloidal solution of gold nanoparticles. Constructing a fluorescence enhancement platform: The pretreated glass substrate was immersed in the above-mentioned gold nanoparticle colloidal solution. Hexane was introduced to form an oil-water interface, and ethanol was injected in a controlled manner to induce the gold nanoparticles to self-assemble into a dense monolayer at the interface. Then, the colloidal suspension was slowly discharged from the bottom of the container, so that the nanoparticle monolayer on the interface was transferred to the glass substrate. Finally, the modified substrate was heat-treated under argon protection: first, the gas was ventilated for 10 minutes, then the temperature was increased to 300 ℃ at 10 ℃ / min and held for 1 hour, and then naturally cooled to room temperature.

[0014] Representative ALSV viral biotinylated single-stranded DNA was selected as the target, such as... Figure 2 As shown in Figure a, the sample was serially diluted from 10,000 pmol to 1 pmol. Complementary single-stranded DNA probes with thiol groups (-SH) modified at the a' end were spotted onto the substrate to capture the corresponding biotinylated single-stranded DNA. ALSV virus standard single-stranded DNA samples of known concentrations, spanning five orders of magnitude, were serially diluted and captured on fluorescently enhanced substrates and glass substrates spotted with complementary single-stranded DNA probes. Images were acquired using a scanner with a 785 nm channel. Figure 2 As shown in Figure b; subsequently, the captured target was detected using biotinylated IRDye800CW-labeled streptavidin. The fluorescence signal of IRDye800CW on the fluorescence enhancement platform exhibited linear changes over a five-order-of-magnitude range, with a correlation coefficient of 0.977. Its detection limit for ALSV single-stranded DNA was as low as 3.91 pmol. Figure 3As shown in Figure c (the detection limit is defined as the blank value plus three standard deviations), the detection method based on a glass substrate failed to achieve effective nucleic acid detection at the same experimental concentration, only detecting fluorescence signals at the highest single-stranded DNA concentration. Compared to the glass substrate, the fluorescence signal intensity of the SA-IRDye800CW fluorescence enhancement platform was enhanced by 50 times at a single-stranded DNA concentration of 10,000 pmol. These results demonstrate that the fluorescence enhancement platform provided in this embodiment can effectively stabilize immobilized DNA probes and achieve high-sensitivity detection over a wide dynamic range. Furthermore, PEF detection analysis was performed using single-stranded DNA of TBEV, SFTSV, MKWV, BJNV, and YEZV viruses. All serially diluted viral samples showed a linear relationship with the detected fluorescence signal, with detection limits ranging from 5.62 to 9.91 pM. Figure 3 The figure shows the slope of the calibration curves for the detection of single-stranded DNA of TBEV, SFTSV, MKWV, BJNV, and YEZV viruses, demonstrating that the fluorescence enhancement platform can achieve comparable detection performance and high detection stability.

[0015] Example 2: Detection and analysis of different single-stranded amplicones by multiple recurrent tick-borne virus detection chips: Source of the virus: In April and May 2024, free ticks were collected using the flag method in Changchun City, Jilin Province, Yichun, Shuangyashan, Daxinganling area of ​​Heilongjiang Province, and Hulunbuir City, Inner Mongolia. The collected tick samples were classified by region and stored in 50 mL centrifuge tubes containing moist cotton balls and grass leaves. The tubes were sealed with gauze and stored at 4°C. The tick samples were removed in batches and placed on ice plates (ticks crawl slowly at low temperatures). Preliminary morphological identification was carried out based on the presence or absence of anal grooves, ossified scutes, scutes mottled, pseudohead position, palps and coxae shape, etc., referring to the "Chinese Economic Insects". The collected tick samples were classified according to region and tick species and aliquoted into 1.5 mL centrifuge tubes, averaging 10 ticks per tube. In a biosafety cabinet, each tube was washed twice with 75% ethanol (to remove bacteria from the tick surface), rinsed twice with DMEM solution, and then 500 μL of DMEM solution and two sterile stainless steel beads were added. The tubes were then placed in an automated rapid sample homogenizer and homogenized at 70 Hz for 90 s. After centrifugation at 12000 rpm for 10 min, the supernatant was collected and aliquoted into two tubes of 140 μL each. One tube was used for DNA extraction for molecular biological identification of the tick species, and the other tube was used for RNA extraction for virus detection. Both tubes were stored at -80℃ for later use. RNA extracted from tick-borne viruses was reverse transcribed into cDNA, and then PCR amplification was performed using 5'-biotin-labeled forward primers and 5'-phosphorylation-modified reverse primers. The primers were designed based on the bilaterally conserved regions of TBEV, SFTSV, MKWV, BJNV, and YEZV viruses (sequences shown in Table 1). The PCR-amplified double-stranded DNA fragments contained a 5'-biotin tag (for subsequent binding to streptavidin-IR800CW) and 3'-phosphate modification (as a phosphatase cleavage site). The amplification product was digested into biotin-labeled single-stranded amplicons using λ exonuclease (which specifically cleaves phosphorylated dsDNA). The obtained biotinylated ssDNA amplicons were co-incubated with a fluorescence-enhanced substrate and identified using SA-IRDye800CW. Table 1

[0016] To achieve high-throughput detection of viral ssDNA, detection probes for TBEV, SFTSV, MKWV, BJNV, and YEZV viruses were immobilized at sites 1-6 in a single reaction region of the fluorescence enhancement platform (e.g., ...). Figure 4 As shown in Table 2, the probe is designed based on the target site sequence and modified with a thiol group (SH) and 12 adenine bases (aaa…) at the 5' end to achieve efficient immobilization via gold-sulfur bonds. Table 2

[0017] After adding SA-IRDye800CW, viral ssDNA was identified by microarray fluorescence signals. Fluorescence imaging and statistical heatmaps of all viral single-stranded amplicon showed that... Figure 5 As shown in Figures b and c, the fluorescence enhancement platform can specifically distinguish different viral nucleic acids without cross-reactivity. Furthermore, PEF detection was performed using diluted cDNA to evaluate its nucleic acid detection sensitivity (12,000 to 1.2 copies per reaction; the concentration of reverse-transcribed cDNA was quantified by droplet digital PCR). The results are shown below. Figure 5 As shown in Figure d, in the detection of representative BJNV virus, the amplified PEF detection can achieve the sensitivity of single-copy cDNA.

[0018] Example 3: Detection efficacy of multiplex tick-borne virus detection chips: Nucleic acid microarrays were prepared in the spotting area. RNA from the test sample was reverse transcribed into cDNA. PCR amplification was performed using 5'-biotin-labeled forward primers and 5'-phosphorylated reverse primers (PCR amplification conditions: denaturation at 98°C for 1 minute; followed by 30 cycles: denaturation at 98°C for 10 seconds, annealing at 55°C for 5 seconds, and extension at 72°C for 30 seconds). The amplification product was digested with λ exonuclease (50 μL reaction system contained 10 μL amplification product, 10 μL λ exonuclease, 5 μL reaction buffer, and 34 μL water, co-incubated at 37°C for 30 minutes, the mixture after reaction was diluted with TE buffer (containing 1 M NaCl) and denatured at 75°C for 10 minutes), generating biotinylated single-stranded amplification products. Detection samples (biotinylated single-stranded amplification products) were added to the detection area for incubation, labeling, and washing. Finally, the average fluorescence intensity obtained by a near-infrared confocal fluorescence scanner reflected the amount of viral nucleic acid bound to each detection site on the substrate. Single-stranded DNA (ssDNA) was detected on a plasma-enhanced fluorescence chip using digested PCR amplified double-stranded DNA fragment products (placed in digestion buffer). The fluorescence intensity of the method in this embodiment was compared with the titer value calculated by qPCR. To obtain a detection signal, the digested product of biotinylated PCR amplification was added to a BSA-blocked fluorescence-enhanced substrate for incubation. After washing with PBS / PBST, IRDye800cw@SA was co-incubated with the biotinylated product to achieve binding. After washing again, the signal was collected using a near-infrared confocal fluorescence scanner, and different viruses were distinguished based on the labeled sites. Statistical heatmaps such as Figure 6 As shown, the fluorescence signals of the test samples exhibited significant intensity differences, clearly distinguishing the nucleic acids of different tick-borne viruses. This indicates that the fluorescence-enhanced substrate platform can effectively identify six tick-borne viruses. Furthermore, the fluorescence intensity distribution at each detection site is plotted as follows: Figure 7 As shown, positive and negative samples can be clearly distinguished. These samples were simultaneously tested using the qPCR method (the target sequence is consistent with the PEF detection region); as Figure 8 As shown in Figures a and b, using qPCR results as the gold standard, PEF detection identified 11 positive samples and 66 negative samples. Based on this, the sensitivity of PEF detection for tick-borne virus nucleic acid reached 91.6%, and the specificity was 100%. It is worth noting that viral titer can characterize its pathogenic potential and can be quantified by qPCR. The correlation coefficient between the fluorescence signal of PEF detection and the viral titer measured by qPCR was 0.661. Figure 8 As shown in Figure c, this further confirms that the embodiments of the present invention have robust performance in the detection and differentiation of tick-borne viruses.

[0019] In summary, this invention provides a low-cost method for fabricating a large-area plasma-enhanced fluorescence substrate with defined gaps on a glass slide. Based on this substrate, six thiol-modified single-stranded nucleic acid probes are loaded to obtain a detection chip. This chip has a detection limit of 5.62 to 9.91 pM for single-stranded nucleic acids and provides a detection dynamic range of up to five orders of magnitude, which is superior to traditional glass substrates. Different single-stranded amplicon samples are identified through differential probes on the chip surface, achieving high-throughput molecular detection. This solves the important technical problem of traditional amplification techniques having an upper limit as the number of pathogens detected increases, and a significant decrease in sensitivity and specificity with increasing target number. It provides an operational platform for the analysis and identification of tick-borne viruses. The substrate prepared by this method utilizes positional information to achieve high-throughput detection, and its significant improvements in multiplexing and cost control are far superior to traditional qPCR, thus showing great potential for application. In addition, fluorescence enhancement can be achieved using nanoparticle films with other structures and materials (such as silver nanoisland films (SIF) and plasma gold glass slides (pGold)), nanopores and nanorod arrays, and nanocavities; or similar enhanced fluorescence substrates can be prepared by other methods such as ultraviolet lithography, electron beam lithography, focused ion beam lithography, chemical deposition, and self-assembly.

[0020] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multi-recurrent tick-borne virus detection chip, characterized in that, The device includes a substrate on which gold nanoparticles are deposited to form a fluorescence enhancement platform. The fluorescence enhancement platform is loaded with six thiol-modified single-stranded nucleic acid probes, which are respectively targeted at tick-borne viruses TBEV, ALSV, SFTSV, MKWV, BJNV, and YEZV, and their sequences are shown in SEQ NO.1-6. The probes are modified with thiol groups and 12 adenine bases at the 5' end.

2. The multi-re-emergence tick-borne virus detection chip according to claim 1, characterized in that, The substrate is a glass substrate.

3. A method for preparing a multi-re-emerging tick-borne virus detection chip as described in claim 1 or 2, characterized in that, Includes the following steps: The glass substrate was pretreated by immersing it in a mixed solution of concentrated H2SO4 and 30% H2O2 for cleaning. Add 1% HAuCl4·3H2O to deionized water, heat to boiling and stir vigorously, then quickly add 2% sodium citrate solution, continue the reaction and cool to room temperature to obtain a gold nanoparticle colloidal solution. The pretreated glass substrate was immersed in a gold nanoparticle ion colloidal solution. Hexane was introduced to form an oil-water interface. Ethanol was injected to promote the self-assembly of gold nanoparticles at the interface to form a dense monolayer. The colloidal suspension was discharged from the bottom, allowing the nanoparticle monolayer on the interface to transfer to the glass substrate. The modified substrate was then heat-treated under argon protection to obtain a fluorescence enhancement platform. Six thiol-modified single-stranded nucleic acid probes were immobilized on a fluorescence enhancement platform. The probes were respectively targeted at tick-borne viruses TBEV, ALSV, SFTSV, MKWV, BJNV, and YEZV, and their sequences are shown in SEQ NO.1-6. The probes were modified with thiol groups and 12 adenine bases at the 5' end.

4. A detection method for a multi-recurrent tick-borne virus detection chip as described in claim 1 or 2, characterized in that, Includes the following steps: The RNA of the test sample was reverse transcribed into cDNA, and PCR amplification was performed using a 5'-biotin-labeled forward primer and a 5'-phosphorylated reverse primer. The amplification product was digested with λ exonuclease, which specifically cleaves phosphorylated double-stranded DNA to produce biotinylated single-stranded amplification products. The biotinylated single-stranded amplification product was incubated on a fluorescence enhancement platform and captured by an immobilized single-stranded nucleic acid probe. It was then detected and identified using a streptavidin-conjugated near-infrared fluorescent dye SA-IRDye800CW, and the signal value was detected using a scanner.

5. The detection method for multiple recurrent tick-borne virus detection chips according to claim 4, characterized in that, The PCR amplification conditions were: denaturation at 98°C for 1 minute; followed by 30 cycles: denaturation at 98°C for 10 seconds, annealing at 55°C for 5 seconds, and extension at 72°C for 30 seconds.

6. The detection method for multiple re-emerging tick-borne virus detection chips according to claim 4, characterized in that, The digestion step using λ exonuclease is as follows: the reaction system contains amplification products, λ exonuclease, reaction buffer and water, and is incubated at 37°C for 30 minutes. The mixture after the reaction is diluted with TE buffer and denatured at 75°C for 10 minutes.

Images