Patent Application
20240102114
The present disclosure relates to the technical field of virus detection, and particularly relates to a proximity ligation assay (PLA)-based detection method for a high-order structure (HOS) of an RNA virus.
Virus is the simplest living organism found so far. Except for prions, the virus is composed of nucleic acids and proteins. Viruses can be divided into RNA viruses and DNA viruses according to the type of nucleic acid. A complete viral nucleic acid is also called a viral genome. The viral genome is a full set of genetic codes of the virus, and the viral genome guides the encoding of all viral proteins and regulates the life cycle of the virus. Recent studies have shown that the viral genome has the function of encoding viral proteins, and fragments of the viral genome can be folded with each other to form a complex spatial structure. This spatial structure (HOS) has great significance for gene coding and the infection and replication of the virus. Therefore, it is of great significance to study the HOS of the viral genome to understand the pathogenicity and the infection and replication of the virus.
Theoretically, identification methods of the HOS of RNA or DNA in cells are suitable for studying the structure of the viral genome. At present, the techniques for studying the HOS of the RNA in the cells can be roughly divided into the following categories: X-ray, nuclear magnetic resonance (NMR), click chemistry and existing PLA. Both the X-ray and the NMR involve high resolution, but have deficiencies of complex technology and inability to study the RNA structures under physiological conditions. These two methods are suitable for fine determination of the structure of RNA complexes. The click chemistry features simplicity and high-throughput, but can only determine whether the RNA is double-stranded and cannot determine the interaction. This method is suitable for the prediction of intracellular RNA structures. The existing PLA is suitable for intracellular RNA structures and interaction, and also has high throughput and RNA structure mapping under physiological conditions. However, this method has complicated operation steps and high requirements for samples, which is not suitable for studying low-level virus samples.
The foundation of studying the HOS of RNA is to study the frequency of spatial contact or interaction between local fragments in RNA molecules. To solve the above problems, researchers have developed a series of research techniques in recent years. A basic idea of these techniques is to fixate RNAs close to each other (by interaction) using an RNA cross-linking agent, treat RNA terminals and ligate interacted RNA fragments, and identify the occurrence frequency of “chimeric” RNAs through high-throughput sequencing and bioinformatics analysis, thereby determining the interaction of RNA fragments. These research methods have played an important role in identifying RNA structures and interactions under different physiological conditions since early invention. All methods in papers published in the earlh stage included the enrichment of cross-linked fragments, such that high requirements on the initial sample size are raised. Generally, at least 20 μg of total RNA was required to meet the experimental needs. However, compared with intracellular RNA, the viral genome has the following characteristics: the viral genome may have a low number of copies and a very low total amount of viral nucleic acid; and the viral genome accounts for a very low number of all host genes. Therefore, the conventional research strategy of RNA structure has many difficulties in studying the structure of the viral genome. In particular, it is difficult to meet the amount of viral nucleic acids required for the experiment, resulting in insufficient analysis coverage, which in turn leads to the loss of a large number of structural details.
In view of this, the purpose of the present disclosure is to provide a PLA-based detection method for a high-order structure (HOS) of an RNA virus. The method can analyze the HOS of the RNA viral genome on low-concentration virus samples, and relatively comprehensive HOS information can be obtained.
The present disclosure provides a PLA-based method for detecting HOS of an RNA virus, which includes the following steps:
1) mixing an RNA virus with a cross-linking agent, conducting cross-linking under ultraviolet (UV) light, and recovering the RNA virus to obtain a cross-linked RNA virus;
2) extracting RNA of the cross-linked RNA virus in step 1);
3) conducting fragmentation on the RNA in step 2) with an RNase III to obtain RNA fragments;
4) ligating the RNA fragments in step 3) and decrosslinking the RNA fragments to obtain decrosslinked RNA fragments;
5) constructing a sequencing library for the decrosslinked RNA fragments in step 4); and
6) conducting high-throughput sequencing on the sequencing library in step 5), and conducting RNA HOS analysis on a sequencing result.
Preferably, the cross-linking agent in step 1) may be a phosphate-buffered saline (PBS) solution containing a psoralen-derived cross-linking agent; and
the psoralen-derived cross-linking agent may have a final concentration of 1-4 μmol/L.
Preferably, the psoralen-based cross-linking agent may include 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT) or EZ-Link™ Psoralen-PEG3-Biotin.
Preferably, the cross-linking agent may further include digitonin with a mass concentration of 0.01-1%.
Preferably, the RNA virus may have a final concentration of 107-109 copies/mL after the mixing in step 1).
Preferably, in step 1), the UV light may have a wavelength of 360-370 nm; and
the cross-linking may last for 15-25 min.
Preferably, a reaction system for the fragmentation with an RNase III in step 3) may include 1 μl of 10×RNase III buffer, 200 ng of RNA and 1 μl of RNase III, supplemented to 20 μl with RNase-free water.
Preferably, the fragmentation with an RNase III may be conducted for 1-10 min at 36-38° C.
Preferably, in step 4), the decrosslinking may be conducted by irradiating the RNA fragments with the UV light;
the UV light may have a wavelength of 250-260 nm; and
the irradiating may last for 1-10 min.
Preferably, the RNA virus may include a coronavirus and a Coxsackie virus.
The present disclosure also provides a construction method of a sequencing library for proximity ligation assay (PLA)-based detection of a high-order structure (HOS) of an RNA virus, including the following steps:
1) mixing an RNA virus with a cross-linking agent, conducting cross-linking under ultraviolet (UV) light, and recovering the RNA virus to obtain a cross-linked RNA virus;
2) extracting RNA of the cross-linked RNA virus in step 1);
3) conducting fragmentation on the RNA extracted in step 2) with RNase III to obtain RNA fragments;
4) ligating the RNA fragments in step 3) and conducting decrosslinking to obtain decrosslinked RNA fragments; and
5) constructing a sequencing library for the decrosslinked RNA fragments in step 4).
Preferably, the cross-linking agent in step 1) is a phosphate-buffered saline (PBS) solution containing a psoralen-derived cross-linking agent; and
the psoralen-derived cross-linking agent has a final concentration of 1-4 μmol/L.
Preferably, the psoralen-based cross-linking agent includes 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT) or EZ-Link™ Psoralen-PEG3-Biotin.
Preferably, the cross-linking agent further includes digitonin with a mass concentration of 0.01-1%.
Preferably, the RNA virus has a final concentration of 107-109 copies/mL after the mixing in step 1).
Preferably, in step 1), the UV light has a wavelength of 360-370 nm; and
the cross-linking lasts for 15-25 min.
Preferably, a reaction system for the fragmentation with RNase III in step 3) includes 1 μl of 10×RNase III buffer, 200 ng of RNA and 1 μl of RNase III, supplementing to 20 μl with RNase-free water.
Preferably, the fragmentation with RNase III is conducted for 1-10 min at 36-38° C.
Preferably, in step 4), the decrosslinking is conducted by irradiating the RNA fragments with the UV light;
the UV light has a wavelength of 250-260 nm; and
the irradiating lasts for 1-10 min.
Preferably, the RNA virus includes a coronavirus and a Coxsackie virus.
The present disclosure provides a PLA-based method for detecting HOS of an RNA virus. In the present disclosure, the RNA virus is cross-linked by UV light under the action of a cross-linking agent, such that the interacted (close) RNA fragments form a covalent bond; on the basis of a relatively low initial sample size, fragmentation is conducted using RNase III nuclease to ensure that each fragmented RNA end is suitable for ligation, which is beneficial to improve the ligation efficiency. The method can also help simplify the operation and reduce the loss of RNA fragments, so that the detection of HOS of RNA viruses is also suitable for low-concentration virus samples. The method provided by the present disclosure is called a high-throughput RNA interaction analysis (Hi-R) method. The Hi-R can map in vivo paired RNA interactions with high sensitivity across the whole genome. In addition, the method provided by the present disclosure can reduce the loss of RNA due to terminal treatment and enrichment of chimeric fragments, thereby making the RNA suitable for direct experiments on small amounts of virus particles. The Hi-R method provided by the present disclosure can be used to map the interaction of fragments and HOS maps in the viral genome, providing a basis for studying structural changes in the life cycle of related viruses and its relationship with biological functions.
The present disclosure provides a PLA-based detection method for HOS of an RNA virus, which includes the following steps:
1) mixing an RNA virus with a cross-linking agent, conducting cross-linking under UV light, and recovering the RNA virus to obtain a cross-linked RNA virus;
2) extracting RNA of the cross-linked RNA virus in step 1);
3) conducting fragmentation on the RNA in step 2) with RNase III to obtain RNA fragments;
4) ligating the RNA fragments in step 3) and conducting decrosslinking to obtain decrosslinked RNA fragments;
5) constructing a sequencing library for the decrosslinked RNA fragments in step 4); and
6) conducting high-throughput sequencing on the sequencing library in step 5), and conducting an RNA HOS analysis on a sequencing result.
In the present disclosure, the RNA virus is mixed with the cross-linking agent, cross-linked under the UV light, and the RNA virus is recovered to obtain the cross-linked RNA virus.
The method is applicable to all types of RNA viruses. In an example of the present disclosure, a specific implementation method is illustrated by taking the coronavirus and the Coxsackie virus as examples.
In the present disclosure, a preparation method of the RNA virus preferably includes infecting cells with an RNA virus, culturing and isolating the RNA virus to obtain RNA virus particles. The infecting is preferably conducted for 20-25 h, more preferably 24 h. The RNA virus has a multiplicity of infection (MOI) of 0.01. The cells have a concentration of 1.0×107-1.0×109/ml.
In the present disclosure, the collected RNA virus mixed with the cross-linking agent has a final concentration of preferably 107-109 copies/mL, more preferably 5×107-5×108 copies/mL. A mixed system has a total volume of preferably 50 μl to 10 ml, more preferably 100 μl. The cross-linking agent is preferably a PBS solution containing a psoralen-derived cross-linking agent. The psoralen-derived cross-linking agent has a final concentration of preferably 1-4 μmol/L, more preferably 2 μmol/L. The psoralen-based cross-linking agent preferably includes AMT or EZ-Link™ Psoralen-PEG3-Biotin. The cross-linking agent preferably further includes digitonin; the digitonin has a mass concentration of preferably 0.01-1%, more preferably 0.01-0.5%. The digitonin as a penetrating agent promotes penetration of the cross-linking agent to reach the RNA through viral capsid protein to improve the cross-linking efficiency.
In the present disclosure, the UV light has a wavelength of preferably 360-370 nm, more preferably 365 nm. The cross-linking is preferably conducted in an ice bath for preferably 5-25 min, more preferably 10-20 min, most preferably 12 min. The cross-linking by UV light facilitates the formation of covalent bonds between the RNA molecules interacting in the virus, and provides convenience for subsequent proximity ligation reaction.
In the present disclosure, there is no specific limitation on a recovery method of the RNA virus, and recovery methods well known in the art can be employed.
In the present disclosure, the RNA of the cross-linked RNA virus is extracted after obtaining the cross-linked RNA virus.
In the present disclosure, there is no specific limitation on the extraction method of the RNA virus, and extraction methods well known in the art can be employed, such as a Trizol method or RNA extraction by an RNeasy Plus Mini Kit (Qiagen).
In the present disclosure, quantitative detection and mass detection of the extracted RNA were preferably conducted. The quantitative detection preferably detects the concentration of the RNA using Qubit to provide guidance for subsequent sampling volume. RNA integrity is preferably detected using Agilent 2100, with a recommended RIN value of greater than 7.
In the present disclosure, the RNA from the RNA virus is preferably detected for UV cross-linking effect. The UV cross-linking effect is preferably detected using a Dotblot kit.
In the present disclosure, fragmentation is conducted on the RNA with RNase III to obtain RNA fragments.
In the present disclosure, a reaction system for the fragmentation with RNase III in step 3) preferably includes 1 μl of 10×RNase III buffer, 200 ng of RNA and 1 μl of RNase III, supplemented to 20 μl with RNase-free water. The fragmentation with RNase III is conducted for preferably 1-10 min, more preferably 2-8 min, and further more preferably 5 min at preferably 36-38° C., more preferably 37° C. The RNA fragments obtained using fragmentation with RNase III can be directly ligated; while RNA fragments obtained using fragmentation with other types of endonucleases, terminals of the RNA fragments need to be subjected to polynucleotide kinase (PNK) treatment before ligation. Therefore, the RNase III enzyme can reduce the experimental steps, reduce the loss of RNA during the experiment and improve the reaction efficiency.
In the present disclosure, the obtained RNA fragments are ligated and decrosslinked to obtain decrosslinked RNA fragments.
In the present disclosure, a reaction system of the ligation includes 20 μl of 10×T4 RNA Ligase buffer, 20 μl of 10 Mm ATP, 1 μl of Superase In, 5 μl of Ribolock RI, 5 μl of T4 RNA Ligase 1 and 200 ng of RNA fragments, supplementing to 200 μl with RNase-free water. The ligation is preferably conducted in a water bath at 16° C. overnight.
The ligated RNA fragments are preferably purified after decrosslinking. In the present disclosure, there is no specific limitation on a purification method, and purification methods well known in the art can be employed, such as recovering trace amounts of RNA using RNeasy Plus Mini Kit RNA (Qiagen).
In the present disclosure, the decrosslinking is preferably conducted by irradiating the RNA fragments with UV light. The UV light preferably has a wavelength of 250-260 nm, more preferably 254 nm. The irradiating preferably lasts for 1-10 min, more preferably 5 min. The cross-linking is preferably conducted in an ice bath. The purpose of the decrosslinking is to destroy the covalent bond to avoid the covalent bond formed by the crosslinking in subsequent library construction from affecting the reverse transcription reaction.
In the present disclosure, a sequencing library is constructed for the decrosslinked RNA fragments.
In the present disclosure, the decrosslinked RNA fragments are preferably detected using Agilent 2100 before sequencing library construction. In the present disclosure, there is no specific limitation on a sequencing library construction method, and sequencing library construction methods well known in the art can be employed, such as SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian User Manual.
In the present disclosure, high-throughput sequencing is conducted on the sequencing library, and an RNA HOS analysis is conducted on a sequencing result.
In the present disclosure, there is no specific limitation on a sequencing method of the high-throughput library, and sequencing methods of the high-throughput library well known in the art can be employed. In the present disclosure, the high-throughput sequencing is completed by entrusting Annoroad Gene Technology Co., Ltd.
In the present disclosure, a chimeric read analysis of the sequencing results preferably refers to the prior art (Travis, A. J., Moody, J., Helwak, A., Tollervey, D., & Kudla, G. (2014)). Hyb: a bioinformatics pipeline for the analysis of CLASH (crosslinking, ligation and sequencing of hybrids) data. Methods, 65(3), 263-273. Doi:10.1016/j.ymeth.2013.10.015).
In the present disclosure, the method can analyze the HOS of an RNA viral genome in supernatant virus particles obtained by cell culture or collection using a high-efficiency proximity ligation. In addition, the method conducts experiments using total RNA initial amount as low as 200 ng, which meets requirements for RNA structural study. Therefore, the method provided by the present disclosure can greatly improve the applicability of the proximity ligation in studies of a structure of microRNAs such as viruses.
For further description, the PLA-based detection method for HOS of an RNA virus provided by the present disclosure is described in detail below with reference to the examples, but these examples may not be understood as limiting the protection scope of the present disclosure.
Application of Hi-R Technology in Analysis of COVID-19 Genome Structure
1. Experimental materials were as follows: Vero cells infected with new coronavirus (SARS-CoV-2), supernatant; cross-linking agent: EZ-Link Psoralen-PEG3-Biotin (Thermo Fisher Scientific) and Permeabilizing agent: digitonin (Sigma-Aldrich).
2. Experimental Steps
2.1. Cross-Linking
9×107/ml of VeroE6 was infected with Wuhan-Hu-1 SARS-CoV-2 virus in a MOI of 0.01 for 24 hours. Three duplicate samples were washed with PBS three times, and the washed cells were collected (denoted as C1, C2 and C3). Remaining infected samples were continued to be cultured for 48 hours, and a virus culture supernatant was mixed with an equal volume of a saturated sodium sulfate solution for 1 hour at 4° C. A mixture was washed with PBS three times and above virus pellets (denoted as V1, V2 and V3) and washed cells (denoted as L1, L2 and L3) were collected. EZ-Link Psoralen-PEG3-Biotin was diluted to 2 μM in a PBS containing 0.01% digitonin, and virus particles or cells were resuspended. After incubating at 37° C. for 10 minutes, the virus particles or cells were spread evenly into one well of a 6-well plate. The 6-well plate was put into a cross-linker after removing cover of the of plate, and cross-linked twice for 10 minutes under 365 nm (the cross-linking machine was placed in a safety cabinet). The 6-well plate was placed on ice during each run of cross-linking. After 10 minutes of cross-linking, the 6-well plate was taken out for new ice replacement, and the cross-linking was continued.
2.2. Extraction of RNA
RNeasy mini kit (Qiagen) was used follow the kit instructions.
2.3. RNA Fragmentation
An RNA fragmentation reaction system was prepared as shown in Table 1 for details.
The reaction system was incubated at 37° C. for 5 minutes, and immediately transferred to RNA purification.
2.4. Purification of Fragmented RNA
Trace RNA was recovered with RNeasy Plus Mini Kit RNA (Qiagen), following the instructions for use.
2.5. Ligation
A ligation system was prepared using agents as shown in Table 2.
The reaction system was mixed evenly in a 16° C. water bath overnight.
2.6. Purification of Ligated RNA
Trace ligated RNA was recovered with RNeasy Plus Mini Kit RNA, following the instructions for use.
2.7. Decrosslinking
The cover of RNase-free EP tube was cut off on an ultra-clean workbench, and the recovered RNA was added to the RNase-free EP tube cover and subjected to 254 nm UV radiation on the ice for 5 min to decrosslink.
2.8. Construction of Sequencing Library
Before library construction, the RNA was detected using Agilent 2100. The library construction referred to SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian User Manual.
2.9. High-Throughput Sequencing and HOS Analysis
The sequencing was conducted with Novaseq 6000, and the sequencing library was provided according to the requirements of a sequencing service provider. The sequencing results was compared with the prior art (Travis, A. J., Moody, J., Helwak, A., Tollervey, D., & Kudla, G. (2014)). Hyb: a bioinformatics pipeline for the analysis of CLASH (crosslinking, ligation and sequencing of hybrids) data. Methods, 65(3), 263-273. Doi:10.1016/j.ymeth.2013.10.015); and RNA HOS analysis of the COVID-19 was conducted.
3. Experimental Results
3.1. Evaluation of Sample Data of Each Group
Results of evaluation of sample data of each group were shown in Table 3.
It can be seen from the above data that the proportion of chimeric fragments in the ligation group is significantly higher than that of a non-ligated control group, and the proportion of chimeric fragments in the ligation group of infected cells is around 10%. The proportion of chimeric fragments ligated to the virus supernatant exceeds 20%, indicating that the RNA compression is relatively tight.
As a control, the ligation efficiency of the COVID-19 genome structure detected using a similar COMRADES method was further analyzed. Results are shown in Table 4.
Determining from the overall detection results in Table 3 and Table 4, the method provided by the present disclosure increases the ratio of chimeric fragments produced by ligation, that is, the effective data ratio is increased. The main reason may be that all terminals are suitable for ligation after RNase III fragmentation to greatly improve the efficiency of ligation.
Meanwhile, in this example, by analyzing the structure of the COVID-19 genome at different life stages, data analysis shows the reliability of the technology, and the details of the internal interaction of the COVID-19 genome can be found. The mechanism of COVID-19 transcription is revealed by analyzing TRS-L-mediated interactions. The similarities and differences of the COVID-19 genome structure in different life states are compared in the details of interaction and the overall structural domain of the genome. Details are as follows.
Application of Hi-R Technology in Analysis of Coxsackie Viral Genome Structure
1. Experimental Materials
Virus particles from the supernatant of HeLa cells infected with Coxsackie virus (CVB-3).
Cross-linking agent: EZ-Link Psoralen-PEG3-Biotin (Thermo Fisher Scientific).
Permeabilizing Agent: Digitonin (Sigma-Aldrich)
2. Experimental Steps
2.1. Crosslinking
1×108/ml of HeLa cells were infected with CVB-3 strain in a MOI of 0.01 for 24 hours. The virus was concentrated by ultracentrifugation. Concentrated virus was filtered with 0.6-μm microporous membrane, transferred to a 38-ml ultrafiltration centrifuge tube, 5 ml of 35% sucrose solution filtered through a 0.2-μm microporous membrane was carefully added to the bottom of the ultrafiltration centrifuge tube. Opening was sealed with soldering iron. Virus particles were centrifuged at 4° C. and 100,000 g for 16 h to the bottom of the tube, upper-layer medium was carefully removed, and the virus particles were collected. The virus particles was resuspended with 100 μl of 2 μM cross-linking agent (containing 0.1% Permeabilizing agent), and incubated at 37° C. for 10 min. The virus particles were spread evenly into one well of a 6-well plate. The 6-well plate was put into a cross-linking machine after removing a cover, and cross-linked twice for 10 minutes at 365 nm. It was required that the cross-linker was placed in a safety cabinet. The 6-well plate was placed on ice during each run of cross-linking. After 10 minutes of cross-linking, the 6-well plate was taken out for replacement of new ice, and the cross-linking was continued. Following the cross-linking, the 6-well plate was taken out and cross-linked virus was treated with 1 ml of Trizol. RNA was extracted using a Trizol method, following the instructions for use.
2.2. Extraction of RNA
RNeasy mini kit (Qiagen) was used following the kit instructions for use.
2.3. RNA Fragmentation
An RNA fragmentation reaction system was prepared using agents as shown in Table 5.
The reaction system was incubated at 37° C. for 5 minutes and immediately transferred to RNA purification.
2.4. Purification of Fragmented RNA
Trace RNA was recovered with RNeasy Plus Mini Kit RNA (Qiagen), following the instructions for use.
2.5. Ligation
A ligation system was prepared using agents as shown in Table 6.
The ligation system was mixed evenly in a 16° C. water bath overnight.
2.6. Purification of Ligated RNA
30 μl Magic Pure RNA Beads (TransGen Biotech)+370 μl of Crowd buffer were added to the ligation system, and mixed well and recovered. Elution was conducted with 15 μl of RNase-free water (Note: if there are too few RNA Beads in this step, the system will be large to affect the adsorption of magnetic beads, and the purification will be very slow). Qubit quantitative analysis was conducted.
2.7. Decrosslinking
The cover of RNase-free EP tube was cut off on an ultra-clean workbench, and the recovered RNA was added to the RNase-free EP tube cover and subjected to 254 nm UV radiation on the ice for 5 min to decrosslink.
2.8. Construction of Sequencing Library
Before library construction, the RNA was detected using Agilent 2100. The library construction referred to SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian User Manual.
2.9. High-Throughput Sequencing
The sequencing was conducted by Hiseq Xten, and the high-throughput sequencing was entrusted to be completed by Annoroad Gene Technology Co., Ltd.
3. Experimental results are shown in Table 7.
The results in Table 7 show that the proportion of chimeric fragments after ligation is much higher than that in the non-ligated group.
The data obtained by sequencing more intuitively shows that the Hi-R technology proposed by the present disclosure can reveal the genomic structural characteristics of the Coxsackie virus CVB13 type, and can be used to compare the structures of two strains of viruses. That is, the intensity of the interaction can be observed, and the domain characteristics of the entire genome can also compared. Details are as follows:
The cross-linking efficiency of Coxsackie virus RNA was determined using a Dotplot method, and the method specifically included following steps: a certain concentration (1 μM or 2 μM) of PBS (containing 0.01% digitonin) of EZ-Link Psoralen-PEG3-Biotin was mixed with a sample of Coxsackie virus particles, and cross-linked under 365 nm of UV light for different time (0, 10 min and 20 min), biotin signals were detected in the sample; a thicker spot indicated a higher cross-linking efficiency. For the Dotplot method, reference can be made to the prior art (Aw, JG, Shen, Y., Wilm, A., Sun, M., Lim, X N, Boon, K L. Wan, Y. (2016). In Vivo Mapping of Eukaryotic RNA Interactomes Reveals Principles of Higher-Order Organization and Regulation. Mol Cell, 62(4), 603-617. doi:10.1016/j.molcel.2016.04.028).
The results are shown in
The above description of examples is merely provided to help understand the method of the present disclosure and a core idea thereof. It should be noted that several improvements and modifications may be made by persons of ordinary skill in the art without departing from the principle of the present disclosure, and these improvements and modifications should also fall within the protection scope of the present disclosure. Various amendments to these embodiments are apparent to those of professional skill in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Thus, the present disclosure is not limited to the examples shown herein but falls within the widest scope consistent with the principles and novel features disclosed herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110447273.0 | Apr 2021 | CN | national |
This patent application is a national stage entry filing under 35 USC 371 to PCT/CN2021/096748 filed on May 28, 2021, which claims the benefit and priority of Chinese Patent Application No. 2021104472730, filed with the China National Intellectual Property Administration (CNIPA) on Apr. 25, 2021, entitled “PROXIMITY LIGATION ASSAY (PLA)-BASED DETECTION METHOD FOR HIGH-ORDER STRUCTURE (HOS) OF RNA VIRUS”, the disclosure of which is incorporated by reference herein in its entirety as part of the present application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/096748 | 5/28/2021 | WO |
