Novel PfAgo protein-mediated EB virus detection system

Abstract

The invention relates to a novel PfAgo protein-mediated EB (epstein-barr) virus detection system, belongs to the technical field of novel virus detection, and aims to solve the problems that early diagnosis of EB viruses is crucial to control and prevention of IM (infantile malnutrition) diseases, and traditional PCR (polymerase chain reaction) has the advantages of being independent of expensive instruments and inconvenient in result reading. Expression and purification of the PfAgo protein; a PfAgo mediated EB virus detection system; preparing plasmids; evaluating specificity and sensitivity; and processing data. The novel PfAgo protein-mediated detection system established in the invention provides a rapid, reliable and sensitive tool for novel field detection and clinical diagnosis of EB virus.

Description

Technical Field

[0001] The present invention relates to the technical field of novel virus detection, and in particular to a novel Epstein-Barr virus detection system mediated by PfAgo protein. Background Art

[0002] Epstein-Barr virus (EBV) belongs to the genus Lymphotropic virus of the family Herpesviridae. It is a double-stranded DNA virus with a genome length of about 173Kb. It is a linear molecule in the virus particle. After entering the infected cell, its DNA is circularized and can replicate itself. EB virus infection is widely distributed in the population, and it is mainly latently infected in B lymphocytes. Once infected, the infected person will carry the virus for life, and the latent virus will relapse when the immunity is low. Oral feeding, kissing, saliva transmission in kindergartens, and droplet transmission of young children can cause children to be infected with EB virus. Among them, one-quarter to three-quarters of EB virus infections will show symptoms of infectious mononucleosis (IM). Although most children with IM can recover on their own or have mild symptoms, some children have low immunity, the body cannot produce enough immune cells to clear the virus, the body is repeatedly attacked by the virus, and the organs are repeatedly damaged, which will eventually lead to organ failure. Therefore, early diagnosis of EB virus is crucial for disease control and prevention in children with IM.

[0003] At present, the diagnosis of EB virus infection mainly includes serology and viral nucleic acid detection. Detection of antibodies in serum is still the cornerstone of diagnosing EB infection, but the serological reaction is relatively complex, and the appearance of antibodies in some children will lag, which adds difficulties to clinical diagnosis. Nucleic acid testing can accurately reflect the infection and load of EB virus, and can distinguish between low-level replication in healthy carriers and high-level active infection in patients with EBV-related diseases, greatly improving the effectiveness of diagnosis. At present, among the various nucleic acid detection methods for EB virus (PCR technology, isothermal amplification technology, CRISPR technology), the most recommended nucleic acid detection method is PCR technology. The advantage of traditional PCR is that it does not rely on expensive instruments, but its results are inconvenient to read. Real-time fluorescence quantitative PCR has high sensitivity and specificity, but it relies on equipment and skilled personnel; Pyrococcus furiosus Argonaute (PfAgo) belongs to the Argonaute protein family and participates in host defense by decomposing invading nucleic acids. It is a DNA-guided programmable enzyme for DNA cleavage with high activity at temperatures between 80°C and 100°C. In recent years, various PfAgo-mediated nucleic acid detection systems have been used for the detection of pathogens such as human papillomavirus, monkeypox virus, and SARS-CoV-2, but the system has not been used for the detection of Epstein-Barr virus. In this study, EBNA1 of Epstein-Barr virus was used as the target gene to establish a detection system based on the combination of traditional PCR and PfAgo. This method has high sensitivity and specificity, and the consistency with real-time fluorescence quantitative PCR detection is 100%, which may be helpful for the clinical diagnosis and molecular epidemiological investigation of Epstein-Barr virus.

[0004] To address the above issues, a new Epstein-Barr virus detection system mediated by PfAgo protein was proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a new Epstein-Barr virus detection system mediated by PfAgo protein. The present method is used to solve the problem that early diagnosis of Epstein-Barr virus is crucial for disease control and prevention in children with IM in the above background. The advantage of traditional PCR is that it does not rely on expensive instruments, but the problem that its results are inconvenient to read.

[0006] To achieve the above object, the present invention provides the following technical solution: a novel Epstein-Barr virus detection system mediated by PfAgo protein, comprising the following steps: S1: DNA extraction; S2: Expression and purification of PfAgo protein; S3: PfAgo-mediated EB virus detection system; S4: plasmid preparation; S5: Specificity and sensitivity evaluation; S6: Data processing.

[0007] Furthermore, samples were obtained from: 20 children in the hospital's pediatric clinic and suspected mononucleosis patients, including 12 males and 8 females, from whom throat swab samples were collected and placed in a collection tube containing 3 mL of sterile saline and the lid was tightened with the specimen number attached; one was tested using the Epstein-Barr virus nucleic acid detection kit (PCR fluorescence method), and the other was stored in an ultra-low temperature freezer at -80°C for future use.

[0008] Furthermore, the main reagents are: 2×PCR reaction mixture; centrifuge tubes and PCR thin-walled tubes; primers, gDNA and molecular beacons; EP01-ordinary agarose gel 6×RNA / DNA loading buffer, EP03-50×TAEBuffer, EP06-GoldenView nucleic acid dye; LDDS2000DNAMarker; Chelex-100 resin; DP204 ordinary DNA product purification kit; Epstein-Barr virus nucleic acid detection kit (fluorescence PCR method).

[0009] Furthermore, the main instruments and equipment are: KP-XW80 vortex mixer; ETC811 gene amplification instrument; Min1524 desktop high-speed centrifuge, ThermoScientific™ digital metal bath; Tanon2500 series fully automatic digital gel image analysis system, EPS-600 digital display constant voltage and constant current electrophoresis instrument, HE-220 high-throughput horizontal electrophoresis tank; -80℃ ultra-low temperature refrigerator and biological safety cabinet.

[0010] Furthermore, the specific steps in S1 are: extracting EB virus genomic DNA from nasopharyngeal swabs by Chelex-100 resin method, adding an appropriate amount of physiological saline to the swab sampling tube, shaking and washing the nasopharyngeal swab, centrifuging at 13000rpm for 1 minute, removing the supernatant and retaining the precipitate; then adding 250μL of Chelex-100, shaking on a vortex shaker for 30s, boiling at 98°C for 10 minutes, and then high-speed centrifuging at 12000rpm for 2min, discarding the precipitate and retaining the supernatant. The supernatant at this time is the required target DNA, which is stored in a -80°C refrigerator.

[0011] Furthermore, the specific steps in S2 are: the PfAgo encoding gene was obtained by gene synthesis and cloned into the pET28a (+) plasmid for recombinant expression; finally, the expression of the His-tag fusion protein was induced in the Escherichia coli BL21 (DE3) strain with 1 mM IPTG at 37°C and purified by Ni affinity chromatography on the AKTA Prime Plus system; the pure protein was stored in storage buffer and aliquots were stored at -80°C.

[0012] Furthermore, the specific steps in S3 are divided into two steps, as follows: S301: PCR and PfAgo lysis; the PCR system was based on previous literature, using forward primer (EBF: 5'-TCATCACCGTCGCTGACTCCGCCATCCAAG-3') and reverse primer (EBR: 5'-GGGTTGGTCCAGGGCCTTCACTTCGGTCTC-3') to amplify the nucleocapsid antigen EB-NA1 gene of the Epstein-Barr virus genome (NC_007605.1); the 25 μL PCR system included 12.5 μL 2× PCR mixture, 2 μL template DNA, 1 μL each of upstream and downstream primers (10 μM) and 8.5 μL H2O; S302: After PCR under optimized conditions (pre-denaturation at 95°C for 1 min, followed by 40 cycles of 95°C for 30 s and 60°C for 30 s, completed within 45 min), the target DNA was cleaved by PfAgo-guided DNA; 20 μL of PfAgo cleavage system contained 2 μL of 10× PfAgo reaction buffer, 0.25 mM to 8 mM MnCl2, dual-labeled 3'-BHQ1 and 5'-FAM-6 reporter probes (concentrations of 0.125 μM to 2 μM) and PfAgo (concentrations of 0.31 μM to 10 μM) were added to 20 μL of PCR product; during the PfAgo cleavage reaction at 95°C, the fluorescence of the system was recorded every 30 min using a Bio-Rad CFX96 instrument.

[0013] Furthermore, the specific steps in S4 are: selecting a conserved region homologous to the EB virus genome nucleocapsid antigen EB-NA1 gene to synthesize a plasmid, and the target fragment contained in the plasmid is 300 bp in size.

[0014] Furthermore, the specific steps in S5 are: using genomic DNA of 7 other pathogens to evaluate the specificity of the established method, including human papillomavirus, varicella-zoster virus, herpes simplex virus, adenovirus, human cytomegalovirus, Haemophilus influenzae type b, Streptococcus pneumoniae and Epstein-Barr virus; selecting a conserved region homologous to the EBNA1 gene to synthesize a plasmid, the sequence range includes the primer amplification region, and the fragment size is 116bp, which is then diluted 10 times to a standard solution of 1 copy / μL to 109 copies / μL to determine the sensitivity evaluation of this method.

[0015] Furthermore, the specific steps in S6 are as follows: All experiments in this study were repeated three times, and the experimental data were analyzed for significant differences using IBM SPSS statistics 22.0 software, with P < 0.05 indicating a significant difference.

[0016] Compared with the prior art, the present invention has the following beneficial effects: A novel Epstein-Barrvirus (EBV) nucleic acid detection method based on the Pyrococcus thermophilus Argonaute (PfAgo) protein was established and evaluated. Multiple pairs of specific PCR primers, guide DNA (gDNA) and molecular beacons were designed by targeting the nucleocapsid antigen EB-NA1 gene of the EBV genome (EBNA1), selecting the best combination and optimizing the reaction conditions. Recombinant plasmids containing the fragment of the EBNA1 gene to be tested were constructed, and the plasmid concentration was gradiently diluted (1-109 copies / μL) and used to evaluate the sensitivity of the method. The genomic DNA of seven pathogens such as human papillomavirus was used as template to evaluate the specificity of the method. Finally, it was used to detect nasopharyngeal swab specimens of children suspected of mononucleosis and verified with real-time fluorescence quantitative PCR. After optimizing the conditions, the lowest detection limit of the method can reach 1 copy / μL. It only amplifies EBV and has no cross-reaction with other pathogens. The detection time is only about 50 minutes, and the results can be observed by naked eyes under ultraviolet or blue light, free from the constraints of professional instruments. By verifying 20 clinical samples suspected of EBV infection, the results were the same as the gold standard fluorescent PCR method; the new PfAgo protein-mediated detection system established in this protocol provides a rapid, reliable and sensitive tool for new on-site detection and clinical diagnosis of EB virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure is the optimal primer combination and the principle diagram of primer amplification; Figure 2 Screening gDNA results for PCR-PfAgo; Figure 3 Screening gDNA results for PCR-PfAgo; Figure 4 Optimize the results of PCR-PfAgo detection conditions; Figure 5 is the sensitivity result of PCR-PfAgo; Figure 6 is the specific result of PCR-PfAgo; Figure 7 It is a flowchart of the operation process of the present invention; Figure 8 This is the specific flow chart of step three. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Example 1: Please refer to Figure 1 and Figure 7-8 ; The novel Epstein-Barr virus detection system mediated by PfAgo protein includes the following steps: S1: DNA extraction; S2: Expression and purification of PfAgo protein; S3: PfAgo-mediated EB virus detection system; S4: plasmid preparation; S5: Specificity and sensitivity evaluation; S6: Data processing.

[0020] Source of samples: 20 children with suspected mononucleosis and pediatric outpatient clinic of Shantou Central Hospital from November 2022 to December 2023 were selected, including 12 males and 8 females. After informed consent from the patients and their guardians, throat swab samples were collected and placed in a collection tube containing 3 mL of sterile saline, the lid was tightened, and the specimen number was attached; one part was tested using the Epstein-Barr virus nucleic acid detection kit (PCR fluorescence method), and the other was stored in a -80°C ultra-low temperature refrigerator for standby use.

[0021] Main reagents: 2×PCR reaction mixture (Beijing Aidelai Biotechnology Co., Ltd.); centrifuge tubes and PCR thin-walled tubes (Shanghai Baisai Biotechnology Co., Ltd.); primers, gDNA and molecular beacons (Suzhou Jinweizhi Biotechnology Co., Ltd.); EP01-ordinary agarose gel 6×RNA / DNA loading buffer, EP03-50×TAEBuffer, EP06-GoldenView nucleic acid dye (Beijing Aidelai Biotechnology Co., Ltd.); LDDS2000DNAMarker (Guangzhou Dongsheng Biotechnology Co., Ltd.); Chelex-100 resin (Bio-Rad Shanghai Co., Ltd.); DP204 ordinary DNA product purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.); Epstein-Barr virus nucleic acid detection kit (fluorescence PCR method) (Aicon Biotechnology (Hangzhou) Co., Ltd.).

[0022] Main instruments and equipment: KP-XW80 vortex mixer (Kaipu Biotechnology Co., Ltd.); ETC811 gene amplification instrument (Dongsheng Innovation Biotechnology Co., Ltd.); Min1524 desktop high-speed centrifuge (Zhuhai Dark Horse Medical Instrument Co., Ltd.), ThermoScientific™ digital metal bath (Thermo Fisher Scientific); Tanon2500 series fully automatic digital gel image analysis system (Shanghai Tianneng Technology Co., Ltd.), EPS-600 digital display constant voltage and constant current electrophoresis instrument, HE-220 high-throughput horizontal electrophoresis tank (Shanghai Tianneng Technology Co., Ltd.); -80℃ ultra-low temperature refrigerator and biological safety cabinet (Qingdao Haier Biomedical Co., Ltd.).

[0023] The specific steps in S1 are: extract Epstein-Barr virus genomic DNA from nasopharyngeal swabs by Chelex-100 resin method, add appropriate amount of physiological saline into the swab sampling tube, shake and wash the nasopharyngeal swab, centrifuge at 13000rpm for 1 minute, remove the supernatant and retain the precipitate; then add 250μL of Chelex-100, shake on a vortex shaker for 30s, boil at 98℃ for 10 minutes, then high-speed centrifuge at 12000rpm for 2min, discard the precipitate and retain the supernatant. The supernatant at this time is the required target DNA, which is stored in a -80℃ refrigerator.

[0024] In this scheme: Best primer screening: All downstream primers (R1, R2, R3) were paired with all upstream primers (F1, F2, F3) for amplification, and the products were purified and electrophoresed on a 2% agarose gel. Figure 1 As shown in A, lanes 1-9 all obtained product bands, but lane 7 was the brightest, with a size of about 116 bp. The amplified fragment was consistent with the theoretical length, and no nonspecific amplification was found. Therefore, we determined that F3R1 was the best RAA primer combination. Figure B shows the principle diagram of primer amplification. Primers were designed in the EBNA-1 gene (11305-97654), and PCR was used to amplify the 166 bp (12387-12502) target fragment; in Figure 1 Middle: A: electrophoresis results of amplification products of each primer combination in RPA reaction, M: DNA marker; 1-9: upstream primer and downstream primer combinations F1R1, F1R2, F1R3, F2R1, F2R2, F2R3, F3R1, F3R2, F3R3. Embodiment 2

[0025] The specific steps in S2 are as follows: according to previous reports of our laboratory [PMID: 38642433], the PfAgo encoding gene was obtained by gene synthesis and cloned into the pET28a (+) plasmid for recombinant expression; finally, 1 mM IPTG was used to induce the expression of His-tag fusion protein in Escherichia coli BL21 (DE3) strain at 37°C and purified by Ni affinity chromatography on the AKTA Prime Plus system (GE Healthcare Life Sciences, Boston, MA); the pure protein was preserved using storage buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.5 mM MnCl2, 15% (v / v) glycerol), and aliquots were stored at −80°C.

[0026] In this protocol: PfAgo detection, recognition and cleavage: Figure 1 As shown, in order to fully demonstrate the recognition and cleavage properties of PfAgo for EBV, we developed a carefully designed detection procedure. First, DNA is extracted from specimens such as swabs, pus or blood from patients suspected of EBV infection. Subsequently, the target region of the EBNA-1 specific gene is amplified using the RCR-PfAgo detection method. After the PfAgo protein binds to the 5'-phosphorylated gDNA, it triggers the specific cleavage of the phosphodiester bond between the 10th and 11th bases of the target DNA (the first cleavage). The first cleavage produces a new 5' phosphorylated secondary gDNA, which provides guidance for PfAgo to perform the second cleavage. In the second cleavage of PfAgo, a single-stranded DNA molecular beacon molecule (MB) is used as a substrate, which emits a fluorescent signal when cleaved. The specific cleavage of the EBNA-1 gene by PfAgo causes BHQ1 to release FAM. In order to deepen the understanding of this basic mechanism, Figure 1 Representative sequences of amplicons, gDNA, secondary cleaved gDNA, and beacon molecules are listed. Embodiment 3

[0027] The specific steps in S3 are divided into two steps, as follows: S301: PCR and PfAgo lysis; the PCR system was based on previous literature, using forward primer (EBF: 5'-TCATCACCGTCGCTGACTCCGCCATCCAAG-3') and reverse primer (EBR: 5'-GGGTTGGTCCAGGGCCTTCACTTCGGTCTC-3') to amplify the nucleocapsid antigen EB-NA1 gene (abbreviated as EBNA1) of the Epstein-Barr virus genome (NC_007605.1); the 25μL PCR system included 12.5μL 2× PCR mixture, 2μL template DNA, 1μL each of upstream and downstream primers (10μM) and 8.5μL H2O; S302: After PCR under optimized conditions (predenaturation at 95°C for 1 min, followed by 40 cycles of 95°C for 30 s and 60°C for 30 s, completed within 45 min), the target DNA was cleaved by PfAgo-guided DNA; 20 μL of the PfAgo cleavage system contained 2 μL of 10× PfAgo reaction buffer, 0.25 mM to 8 mM MnCl2, and dual-labeled 3′-BHQ1 and 5′-FAM-6 reporter probes (at a concentration of 0.125 μM to 2 μM) and PfAgo (at a concentration of 0.31 μM to 10 μM) were added to 20 μL of PCR product; during the 95°C PfAgo cleavage reaction, the fluorescence of the system was recorded every 30 min using a Bio-Rad CFX96 instrument (Bio-Rad, Hercules, CA, USA).

[0028] In this scheme: Best gDNA screening: Each gDNA-probe combination is added to the PfAgo reaction mixture, which contains the following components: Tris / HCl buffer solution, MnCl2, purified PfAgo, 5'-phosphorylated gDNA, ssDNA probe, PCR amplification product and appropriate amount of deionized water. The reaction mixture is incubated at 95°C for 30 minutes; then, the fluorescence intensity of the FAM / ROX signal is recorded every 30 seconds using a fluorescent quantitative PCR instrument. When the fluorescence intensity value reaches the highest platform the fastest, it is the best gDNA-probe combination; Figure 2 As shown in A, the best gDNA combination is: gDNA1-probe1; in Figure 3 Middle: A: Fluorescence curve results of PCR-PfAgo screening for the best gDNA, B: Fluorescence tube diagram under blue transmittance. Embodiment 4

[0029] The specific steps in S4 are: a conserved region homologous to the EB virus genome nucleocapsid antigen EB-NA1 gene (abbreviated as EBNA1) (GenBank accession number: NC_007605.181920-81989) is selected to synthesize a plasmid, and the target fragment contained in the plasmid is 300 bp in size.

[0030] In this scheme: RCR-PfAgo detection system optimization: By adjusting the key components (Mn 2+, probe, and PfAgo) concentrations, an optimization analysis was performed to determine the ideal reaction system for RPA-PfAgo. Initially, different concentrations of PfAgo (4, 4.5, 5.0, 5.5, and 6.0 μM) were evaluated, and it was observed that 4.5 μL PfAgo had the highest enzymatic cleavage efficiency. Then, different concentrations of DNA (2.0, 3.0, 4.0, 5.0, and 6.0 μL) were tested, and the results showed that 3 μL of PfAgo produced the most efficient results. Subsequently, different concentrations of Mn were evaluated. 2+ (0.5, 1.5, 3.0, 4.5 and 6.0 μM), 4.5 μM Mn was observed 2+ The enzyme cleavage efficiency was the highest. Finally, the effects of different probe concentrations on the EBNA-1 gene were studied (10, 25, 40, 55, and 70 μM), and the optimal concentration was found to be 10 μM; Sensitivity analysis of RCR-PfAgo: In order to prepare synthetic plasmid standards for detection using the RCR-PfAgo method, the standards were appropriately diluted, and the plasmid with an initial concentration of 109 copies / μL was diluted 10 times in a gradient, and sensitivity experiments were performed according to the optimal reaction system and reaction conditions. Figure 4 As shown in A, when the plasmid was diluted to a limit of 10 copies / μL, the fluorescence intensity value was still visible, indicating that the minimum detection limit of RCR-PfAgo established in this study was 10 copies / μL, which had a high detection sensitivity; in Figure 4 Middle: A: Fluorescence results of different amounts of PfAgo, B: Fluorescence results of different amounts of DNA, C: Fluorescence results of different amounts of MnCl2, D: Fluorescence results of different concentrations of probes. Embodiment 5

[0031] The specific steps in S5 are as follows: the specificity of the established method was evaluated using genomic DNA of seven other pathogens, including human papillomavirus (HPV), varicella-zostervirus (VZV), herpes simplex virus (HSV), adenovirus (ADV), human cytomegalovirus (HCMV), Haemophilus influenzae type B (Hib), Streptococcus pneumoniae (SP) and Epstein-Barr virus; a conserved region homologous to the EBNA1 gene (GenBank accession number: NC_007605.181920-81989) was selected to synthesize a plasmid, the sequence range included the primer amplification region, and the fragment size was 116 bp, which was then diluted 10 times in a gradient to a standard solution of 1 copy / μL to 109 copies / μL to determine the sensitivity evaluation of this method.

[0032] In this protocol: Analysis of detection specificity of RCR-PfAgo: To evaluate the specificity of the RCR-PfAgo reaction system, the genomes of other pathogens were detected; e.g. Figure 4 As shown in B, it shows that the RCR-PfAgo detection system established in this study is highly specific to the EB virus genome and has no cross-reaction with common pathogens; in Figure 5 Middle: A: PCR-PfAgo reaction results of plasmids with different concentrations, B: fluorescent tube under blue transmittance. Embodiment 6

[0033] The specific steps in S6 are as follows: All experiments in this study were repeated three times, and the experimental data were analyzed for significant differences using IBM SPSS statistics 22.0 software, with P < 0.05 indicating a significant difference.

[0034] In this protocol: Clinical sample test results: This study tested 20 pharyngeal swab samples from children with clinical diagnosis of mononucleosis in pediatrics, of which 18 were positive and 2 were negative. The test results were consistent with those of the fluorescent PCR test method, and there was no statistical difference (P>0.05); in Figure 6 Middle: A: PCR-PfAgo reaction results of different pathogens, B: Agarose gel electrophoresis of different pathogens. Embodiment 7

[0035] Although most people only have mild symptoms after being infected with EB virus, children may experience symptoms such as fever, sore throat, fatigue, etc., and in severe cases, they may also develop complications such as pharyngitis, hepatitis, encephalitis, etc. Therefore, timely and accurate diagnosis of EB virus infection is particularly important. There are many different detection technologies for EB virus, including traditional serological diagnosis methods and molecular diagnosis production methods. Molecular diagnosis methods detect viral DNA or RNA to determine whether a patient is infected with EB virus. It has high sensitivity and good specificity, especially important for early diagnosis of viral infection. PCR is the most commonly used molecular diagnostic method for detecting EB virus infection. Since its introduction in 2006, it has been applied to agriculture, food safety, medicine and transgenic detection. At present, researchers have applied their PCR to various common pathogens in children, such as Coxsackie virus A10, group B streptococcus and enterovirus 71. Ma Xuejun et al. developed the EB virus fluorescent RAA detection technology and combined it with magnetic bead enrichment technology to quickly and specifically detect EB virus in blood within 45 minutes. Although its detection efficiency is basically equivalent to that of qPCR technology, fluorescent RAA detection technology still requires corresponding fluorescent detection instruments, which also limits its application; PfAgo-based technology has been applied to many fields such as food monitoring and agricultural testing. As one of the new nucleic acid detection technologies, PfAgo has potential application prospects. Through the study of the properties of PfAgo protein, a PfAgo-based SARS-CoV-2 detection was developed. Combining PCR technology with PfAgo technology can not only reduce the detection time and improve the sensitivity of PCR, but also eliminate the need for corresponding fluorescence detection instruments. Based on PCR and PfAgo technology, a PCR-PfAgo visualization detection method for Epstein-Barr virus was successfully established using recombinant plasmid and viral genomic DNA as templates. This technology has similar performance to fluorescent PCR technology, with a minimum detection limit of 10 copies / μL. It is simple and fast to operate, and does not require expensive experimental instruments. It has clinical practical significance for the rapid and clear diagnosis of children with Epstein-Barr virus infection. The PCR-PfAgo established in this study is a single-tube closed detection system. After the PCR amplification is completed, there is no need to open the lid and directly enter the next step of PfAgo detection, which avoids cross-contamination caused by amplicons or aerosols in the environment. It is worth noting that the temperature cycle steps of the PCR reaction are complex and time-consuming. In the later stage, it is planned to introduce RPA or LAMP technology to speed up the detection speed and improve the detection efficiency of this technology.

[0036] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0037] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A novel Epstein-Barr virus detection system mediated by PfAgo protein, characterized in that; The following steps are involved: S1: DNA extraction; S2: Expression and purification of PfAgo protein; S3: PfAgo-mediated EB virus detection system; S4: plasmid preparation; S5: Specificity and sensitivity evaluation; S6: Data processing.

2. The novel Epstein-Barr virus detection system mediated by PfAgo protein according to claim 1, characterized in that: Source of samples: Throat swab samples were collected from 20 children in the hospital's pediatric outpatient clinic and suspected mononucleosis patients, including 12 males and 8 females. The samples were placed in a collection tube containing 3 mL of sterile saline and the lid was tightened with the specimen number attached; one sample was tested using the Epstein-Barr virus nucleic acid detection kit (PCR fluorescence method), and the other was stored in a -80°C ultra-low temperature refrigerator for future use.

3. The novel Epstein-Barr virus detection system mediated by PfAgo protein according to claim 2, characterized in that: Main reagents: 2×PCR reaction mixture; centrifuge tubes and PCR thin-walled tubes; primers, gDNA and molecular beacons; EP01-ordinary agarose gel 6×RNA / DNA loading buffer, EP03-50×TAEBuffer, EP06-GoldenView nucleic acid dye; LDDS2000DNAMarker; Chelex-100 resin; DP204 ordinary DNA product purification kit; Epstein-Barr virus nucleic acid detection kit (fluorescence PCR method).

4. The novel Epstein-Barr virus detection system mediated by PfAgo protein according to claim 3, characterized in that: Main instruments and equipment: KP-XW80 vortex mixer; ETC811 gene amplification instrument; Min1524 desktop high-speed centrifuge, ThermoScientific™ digital metal bath; Tanon2500 series fully automatic digital gel image analysis system, EPS-600 digital display constant voltage and constant current electrophoresis instrument, HE-220 high-throughput horizontal electrophoresis tank; -80℃ ultra-low temperature refrigerator and biological safety cabinet.

5. The novel Epstein-Barr virus detection system mediated by PfAgo protein according to claim 4, characterized in that: The specific steps in S1 are: extract Epstein-Barr virus genomic DNA from nasopharyngeal swabs by Chelex-100 resin method, add appropriate amount of physiological saline into the swab sampling tube, shake and wash the nasopharyngeal swab, centrifuge at 13000rpm for 1 minute, remove the supernatant and retain the precipitate; then add 250μL of Chelex-100, shake on a vortex shaker for 30s, boil at 98℃ for 10 minutes, then high-speed centrifuge at 12000rpm for 2min, discard the precipitate and retain the supernatant. The supernatant at this time is the required target DNA, which is stored in a -80℃ refrigerator.

6. The novel Epstein-Barr virus detection system mediated by PfAgo protein according to claim 5, characterized in that: The specific steps in S2 are as follows: the PfAgo encoding gene was obtained by gene synthesis and cloned into the pET28a (+) plasmid for recombinant expression; finally, the expression of the His-tag fusion protein was induced in the Escherichia coli BL21 (DE3) strain with 1 mM IPTG at 37°C and purified by Ni affinity chromatography on the AKTA Prime Plus system; the pure protein was preserved using storage buffer and aliquots were stored at -80°C.

7. The novel Epstein-Barr virus detection system mediated by PfAgo protein according to claim 6, characterized in that: The specific steps in S3 are divided into two steps, as follows: S301: PCR and PfAgo lysis; the PCR system was based on previous literature, using forward primer (EBF: 5'-TCATCACCGTCGCTGACTCCGCCATCCAAG-3') and reverse primer (EBR: 5'-GGGTTGGTCCAGGGCCTTCACTTCGGTCTC-3') to amplify the nucleocapsid antigen EB-NA1 gene of the Epstein-Barr virus genome (NC_007605.1); the 25 μL PCR system included 12.5 μL 2× PCR mixture, 2 μL template DNA, 1 μL each of upstream and downstream primers (10 μM) and 8.5 μL H2O; S302: After PCR under optimized conditions (pre-denaturation at 95°C for 1 min, followed by 40 cycles of 95°C for 30 s and 60°C for 30 s, completed within 45 min), the target DNA was cleaved by PfAgo-guided DNA; 20 μL of PfAgo cleavage system contained 2 μL of 10× PfAgo reaction buffer, 0.25 mM to 8 mM MnCl2, dual-labeled 3'-BHQ1 and 5'-FAM-6 reporter probes (concentrations of 0.125 μM to 2 μM) and PfAgo (concentrations of 0.31 μM to 10 μM) were added to 20 μL of PCR product; during the PfAgo cleavage reaction at 95°C, the fluorescence of the system was recorded every 30 min using a Bio-Rad CFX96 instrument.

8. The novel Epstein-Barr virus detection system mediated by PfAgo protein according to claim 7, characterized in that: The specific steps in S4 are: selecting a conserved region homologous to the EB virus genome nucleocapsid antigen EB-NA1 gene to synthesize a plasmid, and the target fragment contained in the plasmid is 300 bp in size.

9. The novel PfAgo protein-mediated Epstein-Barr virus detection system according to claim 8, characterized in that: The specific steps in S5 are as follows: the specificity of the established method was evaluated using genomic DNA of seven other pathogens, including human papillomavirus, varicella-zoster virus, herpes simplex virus, adenovirus, human cytomegalovirus, Haemophilus influenzae type b, Streptococcus pneumoniae and Epstein-Barr virus; a conserved region homologous to the EBNA1 gene was selected to synthesize a plasmid with a sequence range covering the primer amplification region and a fragment size of 116 bp, which was then diluted 10-fold to a standard solution of 1 copy / μL to 109 copies / μL to determine the sensitivity of the method.

10. The novel Epstein-Barr virus detection system mediated by PfAgo protein according to claim 9, characterized in that: The specific steps in S6 are as follows: All experiments in this study were repeated three times, and the experimental data were analyzed for significant differences using IBM SPSS statistics 22.0 software, with P < 0.05 indicating a significant difference.

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