Probe combinations for in situ detection of negative-strand RNA viruses and differentiation of viral vRNA, cRNA, and mRNA, and their applications.

Abstract

This invention discloses a probe combination for in situ detection of negative-sense RNA viruses and differentiation of viral vRNA, cRNA, and mRNA, and its application. The probe combination includes probe P1, probe P2, and probe P3; probe P1 is a viral RNA type identification probe and also serves as a primer for rolling circle amplification; probes P2 and P3 are RNA sequence origin recognition probes. When probes P1, P2, and P3 work synergistically on the same target RNA, probes P2 and P3, guided by probe P1, form two ligation-aligned gaps on the target RNA. After ligation, a single-stranded closed circular DNA is formed. Using probe P1 as a primer, rolling circle amplification is performed, generating a detectable fluorescent signal.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, specifically to a probe combination for in situ detection of negative-strand RNA viruses and differentiation of viral vRNA, cRNA and mRNA, and its application. Background Technology

[0002] Negative-sense RNA viruses (–ssRNA viruses) replicate using RNA-dependent RNA polymerase. They exhibit high mutation rates, short replication cycles, and strong cross-host capability, encompassing important pathogens from the Paramyxoviridae, Filoviridae, Bunyavirales, and Orthomyxoviridae families. They have long been a major cause of large-scale respiratory and hemorrhagic fever epidemics. The high variability (compared to segmented viruses) and gene reassortment of negative-sense RNA viruses make them prone to immune evasion and drug resistance, thus increasing the demand for precise detection technologies with spatiotemporal resolution.

[0003] Represented by the influenza virus, which belongs to the Orthomyxoviridae family, it is an enveloped, segmented, negative-sense RNA virus, classified into types A, B, C, and D. Type A influenza virus has the widest host range. Influenza virus replication involves three different forms of RNA: viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA). vRNA is the virus's genetic material, composed of eight segments: PB2, PB1, PA, HA, NP, NA, M, and NS, encapsulated by the nucleoprotein NP, forming the ribonucleoprotein complex (RNP). Once inside the cell nucleus, vRNP is transcribed to synthesize viral mRNA, which is then translated to synthesize viral proteins. During viral genome replication, cRNA is first synthesized using vRNA as a template, and then progeny vRNA is synthesized using cRNA as a template. The 13 bases at the 5' end and the 12 bases at the 3' end of the vRNA are highly conserved; this region forms the influenza virus promoter. Both cRNA and mRNA are synthesized using vRNA as a template. The cRNA sequence is inversely complementary to the vRNA sequence, and the mRNA sequence is completely identical to the cRNA except for the 5' cap and the 3' poly(A) tail.

[0004] Distinguishing between vRNA, cRNA, and mRNA of negative-sense RNA viruses has long been a technical challenge, and preserving the spatial distribution information of RNA is even more difficult. The earliest primer extension method used involved binding specific primers for the three viral RNAs to their corresponding target RNAs and extending the RNA under radiolabeled conditions to reveal the various distributions of the three RNAs. However, this method suffered from low sensitivity and radioactive contamination. Northern blotting separated RNA samples using agarose gel electrophoresis and transferred them to a membrane, then hybridized them with labeled probes to the target RNA. This method could clearly identify the size, integrity, and abundance of different types of RNA, but it was complex and had low sensitivity. RT-qPCR used specific primers to reverse transcribe specific RNAs, and then monitored the amplification process with real-time fluorescence signals. This method could specifically distinguish and quantify vRNA, cRNA, and mRNA with high precision and sensitivity, but had low throughput. In recent years, RNA-seq has shown advantages such as high throughput and low primer bias in distinguishing and detecting the three viral RNAs. However, all of the above methods require lysing the sample to extract RNA, which destroys cell morphology and fails to preserve spatial information. Fluorescence in situ hybridization (FISH) can accurately reproduce the expression state and spatial distribution of RNA in cells, making it an excellent technique for studying viral RNA.

[0005] However, current fluorescence in situ hybridization techniques cannot distinguish between vRNA, cRNA, and mRNA of negative-sense RNA viruses. The core challenge lies in differentiating between cRNA and mRNA, two important viral RNAs. Taking influenza virus as an example, its mRNA and cRNA are highly similar, inevitably leading to mismatches between the two when conventional probes identify one, generating erroneous signals. This results in existing studies involving RNA spatial localization often treating them interchangeably. To distinguish the three types of influenza virus RNA, the 3' end sequence of the RNA is needed; however, it should be noted that this 3' region is relatively conserved across different gene segments of the influenza virus. To achieve this goal, the following two sub-goals need to be accomplished simultaneously: distinguishing vRNA, cRNA, and mRNA based on the RNA 3' end sequence; and simultaneously, identifying viral gene-specific sequences to determine which segment of the virus the RNA originates from.

[0006] In summary, achieving in-situ visualization of the three negative-sense viral RNAs remains a major technical challenge. There is an urgent need to develop new FISH techniques to improve the sensitivity and specificity of viral RNA detection, particularly for in-situ visualization of the three viral RNAs. This is of great significance for comprehensively revealing the complex regulatory networks of viruses from the genome to proteins, antiviral drug development and vaccine design, disease diagnosis, and the formulation of antiviral strategies. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a probe combination for in situ detection of negative-sense RNA viruses and differentiation of viral vRNA, cRNA, and mRNA, along with its applications. This invention proposes a novel probe design method and fluorescence in situ hybridization (FISH) procedure. Specificity is ensured through asymmetric thermodynamic structure with three probes, dual-round hybridization, and double-linked circularization. Signal amplification and visualization are achieved through rolling circle amplification (RCA). This method eliminates the need for in situ reverse transcription and can differentiate and visualize the vRNA, cRNA, and mRNA of negative-sense RNA viruses in various experimental systems, including infected cells and tissue sections.

[0008] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a probe combination for in situ detection of negative-strand RNA viruses and differentiation of viral vRNA, cRNA and mRNA, wherein the probe combination includes any one or more combinations of probe combination 1 to probe combination 9. The probe combination 1 includes probes P1-vRNA-HA, P2-vRNA-HA, and P3-vRNA-HA for detecting viral vRNA, probes P1-cRNA-HA, P2-cRNA-HA, and P3-cRNA-HA for detecting cRNA, and probes P1-mRNA-HA, P2-mRNA-HA, and P3-mRNA-HA for detecting mRNA. The probe combination 2 includes probes P1-vRNA-M, P2-vRNA-M, and P3-vRNA-M for detecting viral vRNA, probes P1-cRNA-M, P2-cRNA-M, and P3-cRNA-M for detecting cRNA, and probes P1-mRNA-M, P2-mRNA-M, and P3-mRNA-M for detecting mRNA. The probe combination 3 includes probes P1-vRNA-NA, P2-vRNA-NA, and P3-vRNA-NA for detecting viral vRNA, probes P1-cRNA-NA, P2-cRNA-NA, and P3-cRNA-NA for detecting cRNA, and probes P1-mRNA-NA, P2-mRNA-NA, and P3-mRNA-NA for detecting mRNA. The probe combination 4 includes probes P1-vRNA-NP, P2-vRNA-NP, and P3-vRNA-NP for detecting viral vRNA, probes P1-cRNA-NP, P2-cRNA-NP, and P3-cRNA-NP for detecting cRNA, and probes P1-mRNA-NP, P2-mRNA-NP, and P3-mRNA-NP for detecting mRNA. The probe combination 5 includes probes P1-vRNA-NS, P2-vRNA-NS, and P3-vRNA-NS for detecting viral vRNA, probes P1-cRNA-NS, P2-cRNA-NS, and P3-cRNA-NS for detecting cRNA, and probes P1-mRNA-NS, P2-mRNA-NS, and P3-mRNA-NS for detecting mRNA. The probe combination 6 includes probes P1-vRNA-PA, P2-vRNA-PA, and P3-vRNA-PA for detecting viral vRNA, probes P1-cRNA-PA, P2-cRNA-PA, and P3-cRNA-PA for detecting cRNA, and probes P1-mRNA-PA, P2-mRNA-PA, and P3-mRNA-PA for detecting mRNA. The probe combination 7 includes probes P1-vRNA-PB1, P2-vRNA-PB1, and P3-vRNA-PB1 for detecting viral vRNA, probes P1-cRNA-PB1, P2-cRNA-PB1, and P3-cRNA-PB1 for detecting cRNA, and probes P1-mRNA-PB1, P2-mRNA-PB1, and P3-mRNA-PB1 for detecting mRNA. The probe combination 8 includes probes P1-vRNA-PB2, P2-vRNA-PB2, and P3-vRNA-PB2 for detecting viral vRNA, probes P1-cRNA-PB2, P2-cRNA-PB2, and P3-cRNA-PB2 for detecting cRNA, and probes P1-mRNA-PB2, P2-mRNA-PB2, and P3-mRNA-PB2 for detecting mRNA. The probe combination 9 includes probes P1-vRNA, P2-vRNA, and P3-vRNA for detecting viral vRNA, probes P1-cRNA, P2-cRNA, and P3-cRNA for detecting cRNA, and probes P1-mRNA, P2-mRNA, and P3-mRNA for detecting mRNA. The probe P1 is a viral RNA type identification probe and also serves as a primer for rolling circle amplification. Probe P1 contains an RNA target complementary region, a probe P2 complementary region, and a probe P3 complementary region. The target site of the RNA target complementary region is any one of the internal sequence of vRNA, the conserved 3′ end sequence of cRNA, and the poly(A) tail region of mRNA immediately adjacent to the coding region. The probe P2 is an RNA sequence origin recognition probe, comprising an RNA binding arm A and a P1 complementary arm A; the RNA binding arm A is complementary to the adjacent internal sequence of the target site of the RNA target complementary region on the target RNA, and the P1 complementary arm A is complementary to the P2 complementary region of the probe P1; the RNA binding arm A on the probe P2 is short while the P1 complementary arm A is long. The probe P3 is an RNA sequence source recognition probe, comprising an RNA binding arm B, a fluorescent detection probe binding sequence, and a P1 complementary arm B; the RNA binding arm B is complementary to the adjacent internal sequence of the complementary sequence of the RNA binding arm A on the target RNA, and the P1 complementary arm B is complementary to the P3 complementary region of the probe P1; the RNA binding arm B on the probe P3 is long while the P1 complementary arm B is short. When probes P1, P2, and P3 work synergistically on the same target RNA, probes P2 and P3, guided by probe P1, form two ligation gaps aligned end-to-end on the target RNA. After ligation, a single-stranded closed circular DNA is formed. Using probe P1 as a primer, rolling circle amplification is performed, generating a detectable fluorescent signal. The probes P1-vRNA-HA, P1-vRNA-M, P1-vRNA-NA, P1-vRNA-NP, P1-vRNA-NS, P1-vRNA-PA, P1-vRNA-PB1, P1-vRNA-PB2, P1-cRNA-HA, P1-cRNA-M, P1-cRNA-NA, P1-cRNA-NP, P1-cRNA-NS, P1-cRN The nucleotide sequences of A-PA, P1-cRNA-PB1, P1-cRNA-PB2, P1-mRNA-HA, P1-mRNA-M, P1-mRNA-NA, P1-mRNA-NP, P1-mRNA-NS, P1-mRNA-PA, P1-mRNA-PB1, P1-mRNA-PB2, P1-vRNA, P1-cRNA, and P1-mRNA are as shown in SEQ. ID NO: 1~SEQ ID NO: 27; The nucleotide sequences of the probes P2-vRNA-HA, P2-vRNA-M, P2-vRNA-NA, P2-vRNA-NP, P2-vRNA-NS, P2-vRNA-PA, P2-vRNA-PB1, P2-vRNA-PB2, P2-cRNA-HA, P2-cRNA-M, P2-cRNA-NA, P2-cRNA-NP, P2-cRNA-NS, P2-cRNA-PA, P2-cRNA-PB1, P2-cRNA-PB2, P2-mRNA-HA, P2-mRNA-M, P2-mRNA-NA, P2-mRNA-NP, P2-mRNA-NS, P2-mRNA-PA, P2-mRNA-PB1, P2-mRNA-PB2, P2-vRNA, P2-cRNA, and P2-mRNA are shown in SEQ ID NO: 28 to SEQ ID NO: 54, respectively. The probes P3-vRNA-HA, P3-vRNA-M, P3-vRNA-NA, P3-vRNA-NP, P3-vRNA-NS, P3-vRNA-PA, P3-vRNA-PB1, P3-vRNA-PB2, P3-cRNA-HA, P3-cRNA-M, P3-cRNA-NA, P3-cRNA-NP, P3-cRNA-NS, P3-cRN The nucleotide sequences of A-PA, P3-cRNA-PB1, P3-cRNA-PB2, P3-mRNA-HA, P3-mRNA-M, P3-mRNA-NA, P3-mRNA-NP, P3-mRNA-NS, P3-mRNA-PA, P3-mRNA-PB1, P3-mRNA-PB2, P3-vRNA, P3-cRNA, and P3-mRNA are as shown in SEQ. ID NO:55~SEQ ID NO:81.

[0009] Further, the RNA target complementary region consists of U bases, where U is 14–30 bases; the P2 complementary region and P1 complementary arm A each consist of V bases, where V is 12–24 bases; the P3 complementary region and P1 complementary arm B each consist of W bases, where W is less than V, and when V is even, W is V / 2, and when V is odd, W is (V+1) / 2; the RNA binding arm A consists of X bases, where X is 6–12 bases; the RNA binding arm B consists of Y bases, where Y is twice X; and the fluorescent detection probe binding sequence consists of 30–36 bases.

[0010] Furthermore, the fluorescent detection probes built into the probe P3 targeting different RNAs have different binding sequences, and the fluorescent molecules carried by each fluorescent detection probe have a fluorescence emission spectrum that is different from that of the other fluorescent detection probes, so as to achieve simultaneous multicolor differentiation detection of multiple RNAs; the virus is any one of influenza virus, respiratory syncytial virus, rabies virus and filovirus.

[0011] Furthermore, the probe combination is suitable for in situ differentiation and detection of influenza virus vRNA, cRNA, and mRNA.

[0012] The present invention also provides a kit for in situ detection of negative-strand RNA viruses and differentiation of viral vRNA, cRNA and mRNA, the kit comprising the probe combination described above.

[0013] The present invention also provides an application of the probe combination or the kit described herein, the application including any one or more of the following: (a) In situ differentiation detection of influenza virus vRNA, cRNA and mRNA; (b) In situ detection of vRNA, cRNA and mRNA of respiratory syncytial virus, rabies virus or filovirus; (c) In situ detection of single-base point mutations in the genome of negative-strand RNA viruses; (d) In situ detection of defective interfering RNA generated during viral replication.

[0014] This invention also provides a method for in situ detection of negative-strand RNA viruses and differentiation of viral vRNA, cRNA, and mRNA using the aforementioned kit, comprising the following steps: (1) The 5′ ends of probe P2 and probe P3 were phosphorylated using T4 polynucleotide kinase, respectively; probe P1 and phosphorylated probe P2 were incubated and then annealed to form probe P1 / P2 complex. (2) The samples to be tested were sequentially fixed, dehydrated, and permeabilized; (3) Add the prepared probe P1 / P2 complex to the pretreated sample and incubate it to allow probe P1 to pair with the RNA type site on the target RNA and guide probe P2 to the corresponding target site; wash away non-specifically bound probe P2. (4) Add phosphorylated probe P3 to align and pair it with the segment-specific internal sequence on the target RNA and the probe P1 / P2 complex. Probe P2 and probe P3 form two ligation gaps that are aligned end to end. Wash away non-specifically bound probe P3. (5) Using SplintR ligase and T4 DNA ligase, probe P2 and probe P3 are joined at the two gaps, forming a single-stranded closed circular DNA. (6) Using probe P1 as a primer and the single-stranded closed circular DNA formed in step (5) as a template, rolling circle amplification was performed using Phi29 DNA polymerase to generate amplification products. (7) Based on the fluorescent detection probe binding sequence of probe P3, a fluorescent detection probe fp is designed. The fluorescent detection probe fp is hybridized with the fluorescent detection probe binding sequence pre-placed by probe P3 on the amplification product to obtain a visible fluorescent signal. Using a confocal microscopy system for imaging, different types of RNA exhibit different fluorescent signals, thus enabling in situ detection and differentiation of three types of viral RNA.

[0015] Furthermore, in step (1), the incubation conditions are 95~98℃ for 2~3 minutes, and the annealing treatment is specifically: cooling down to 25~30℃ at a cooling rate of 0.2℃ per second; In step (2), the fixation solution is 4% paraformaldehyde; the dehydration solution is 100% methanol; the permeation solution is 0.1 M hydrochloric acid solution; for tissue section samples, the permeation treatment also includes treatment with pepsin before hydrochloric acid treatment.

[0016] Furthermore, in steps (3) and (4), the pairing conditions are overnight incubation at 36~38℃ for 8~12 hours; In step (5), the connection reaction conditions are: first react at 16°C for 1 hour, then react at 25°C for 1 hour; In step (6), the reaction conditions for rolling circle amplification are 30°C for 2 hours. In step (7), the hybridization conditions are incubation at 25°C for 3 hours.

[0017] Furthermore, when probe P3 is P3-vRNA-HA, the corresponding fluorescent detection probe fp is fp-vRNA-HA; when probe P3 is P3-vRNA-M, the corresponding fluorescent detection probe fp is fp-vRNA-M; when probe P3 is P3-vRNA-NA, the corresponding fluorescent detection probe fp is fp-vRNA-NA; when probe P3 is P3-vRNA-NP, the corresponding fluorescent detection probe fp is fp-vRNA-NP; when probe P3 is P3-vRNA-NS, the corresponding fluorescent detection probe fp is fp-vRNA-NS; and when probe P3 is P3-vRNA-PA, the corresponding fluorescent detection probe fp is fp-vRNA-NA. When probe P3 is fp-vRNA-PA; when probe P3 is P3-vRNA-PB1, the corresponding fluorescent detection probe fp is fp-vRNA-PB1; when probe P3 is P3-vRNA-PB2, the corresponding fluorescent detection probe fp is fp-vRNA-PB2; when probe P3 is P3-cRNA-HA, the corresponding fluorescent detection probe fp is fp-cRNA-HA; when probe P3 is P3-cRNA-M, the corresponding fluorescent detection probe fp is fp-cRNA-M; when probe P3 is P3-cRNA-NA, the corresponding fluorescent detection probe fp is fp-cRNA-NA; when probe P3 is P3-cRNA-NP, the corresponding fluorescent detection probe fp is fp-cRNA-NA; when probe P3 is P3-cRNA-NP, the corresponding fluorescent detection probe fp is fp-cRNA-NA. The corresponding fluorescent detection probe fp is fp-cRNA-NP; when probe P3 is P3-cRNA-NS, the corresponding fluorescent detection probe fp is fp-cRNA-NS; when probe P3 is P3-cRNA-PA, the corresponding fluorescent detection probe fp is fp-cRNA-PA; when probe P3 is P3-cRNA-PB1, the corresponding fluorescent detection probe fp is fp-cRNA-PB1; when probe P3 is P3-cRNA-PB2, the corresponding fluorescent detection probe fp is fp-cRNA-PB2; when probe P3 is P3-mRNA-HA, the corresponding fluorescent detection probe fp is fp-mRNA-HA; when probe P3 is P... When P3 is P3-mRNA-M, the corresponding fluorescent detection probe fp is fp-mRNA-M; when P3 is P3-mRNA-NA, the corresponding fluorescent detection probe fp is fp-mRNA-NA; when P3 is P3-mRNA-NP, the corresponding fluorescent detection probe fp is fp-mRNA-NP; when P3 is P3-mRNA-NS, the corresponding fluorescent detection probe fp is fp-mRNA-NS; when P3 is P3-mRNA-PA, the corresponding fluorescent detection probe fp is fp-mRNA-PA; when P3 is P3-mRNA-PB1, the corresponding fluorescent detection probe fp is fp-mRNA-PB1.When probe P3 is P3-mRNA-PB2, the corresponding fluorescent detection probe fp is fp-mRNA-PB2; when probe P3 is P3-vRNA, the corresponding fluorescent detection probe fp is fp-vRNA; when probe P3 is P3-cRNA, the corresponding fluorescent detection probe fp is fp-cRNA; when probe P3 is P3-mRNA, the corresponding fluorescent detection probe fp is fp-mRNA. The nucleotide sequences of the fluorescent detection probes fp-vRNA-HA, fp-vRNA-M, fp-vRNA-NA, fp-vRNA-NP, fp-vRNA-NS, fp-vRNA-PA, fp-vRNA-PB1, fp-vRNA-PB2, fp-cRNA-HA, fp-cRNA-M, fp-cRNA-NA, fp-cRNA-NP, fp-cRNA-NS, fp-cRNA-PA, fp-cRNA-PB1, fp-cRNA-PB2, fp-mRNA-HA, fp-mRNA-M, fp-mRNA-NA, fp-mRNA-NP, fp-mRNA-NS, fp-mRNA-PA, fp-mRNA-PB1, fp-mRNA-PB2, fp-vRNA, fp-cRNA, and fp-mRNA are shown in SEQ ID NO: 82 to SEQ ID NO: 108, respectively.

[0018] The principle of this invention: 1. The overall framework of the method of this invention is as follows: asymmetric thermodynamic three-probe combination with dual-round hybridization and dual-ligation to form a circular structure, followed by rolling circle amplification (RCA) to amplify the signal. Only when both RNA type recognition and RNA segment origin recognition conditions are simultaneously and precisely met can the three probes converge on the target RNA, be catalyzed by ligase to form a closed single-stranded DNA circular structure, and then initiate rolling circle amplification, generating fluorescent signal spots. If either condition is not met, the probes cannot form a circular structure and no signal is generated, fundamentally suppressing false positives caused by mismatches.

[0019] 2. In the three-probe system, probe P1 is an RNA type identification probe. Its target site is selected based on the unique characteristic regions of the three RNAs: it binds to the internal sequence when detecting vRNA; it binds to the conserved 3' end sequence (i.e., the influenza virus replication promoter region, which is distinct from mRNA) when detecting cRNA; and it binds to the specific region of the poly(A) tail adjacent to the coding region when detecting mRNA. Probe P1 also serves as a primer for rolling circle amplification. Its structure also contains segments complementary to probes P2 and P3, respectively, spatially guiding probes P2 and P3 to their corresponding positions on the target RNA.

[0020] 3. Probes P2 and P3 work together to identify the segment origin of RNA, employing an asymmetric thermodynamic structure design. Probe P2 has a shorter RNA-binding arm A and a longer complementary arm A of P1; probe P3 is the opposite, with a longer RNA-binding arm B and a shorter complementary arm B of P1. This asymmetric design is based on the fact that the short arm of probe P2 is extremely sensitive to base mismatches. If the target sequence is not completely complementary, the short arm binding is unstable and will detach during the washing step, eliminating non-specific binding. The long arm of probe P3 provides sufficient binding stability for the second round of hybridization, ensuring precise probe anchoring. Probe P3 contains a centrally located fluorescent detection probe binding sequence. Different target RNAs carry different internal sequences on probe P3, corresponding to different fluorescence channels, achieving multi-color differentiation.

[0021] 4. The circularization step employs a combination of Splint R ligase and T4 DNA ligase, catalyzing ligation at the two gaps on probes P2 and P3 respectively. Splint R ligase has single-base resolution for base matching at the gaps; if any gap contains a base mismatch, ligation cannot be completed, thus blocking circularization and subsequent amplification. This dual-ligation design constitutes a strict AND gate logic: all three probes must simultaneously and precisely hybridize with the correct target, and there must be no mismatches at either gap for circularization to occur and amplification to begin, significantly suppressing non-specific signals.

[0022] 5. After circularization, Phi29 DNA polymerase uses probe P1 as a primer and the closed DNA loop as a template to continuously perform rolling circle amplification, generating amplification products (RCPs) containing a large number of tandem repeat sequences in situ. The RCPs coil and anchor at the location of the target RNA. After the fluorescently labeled detection probe hybridizes with the RCPs, it produces bright and discrete fluorescent signal spots, which can be clearly imaged under a conventional confocal microscope for single-cell copy number quantification.

[0023] The beneficial effects of this invention are: 1. This invention achieves, for the first time, in situ visualization of influenza virus vRNA, cRNA, and mRNA simultaneously in a single cell: the type of cRNA or mRNA is distinguished by probe P1, and the genome segment from which the RNA originates is determined by probes P2 and P3, overcoming the long-standing limitation of FISH methods in being unable to distinguish the three types of influenza virus RNA.

[0024] 2. This invention can be extended to the in situ detection of three types of RNA from other negative-strand RNA viruses.

[0025] 3. This invention proposes a highly specific FISH technique: through three asymmetric thermodynamic structure probes, two rounds of hybridization, and two connection reactions, all of which must be correct to obtain a template for signal amplification and visualization, which greatly suppresses non-specific signals.

[0026] 4. The probe of this invention adopts an asymmetric length design (short / long arm combination), which improves the probability of mismatch dissociation and significantly reduces background. It can detect and distinguish influenza virus vRNA, cRNA and mRNA in multiple scenarios such as cell samples, tissue samples and transfected mini-genome samples. It is not affected by host DNA and RNA interference and has extremely low non-specific signal. 5. This invention utilizes two enzymes, SplintR ligase and T4 DNA ligase, in the ligation step, making it more specific and efficient; 6. This invention directly targets viral RNA without in situ reverse transcription, simplifying the process, improving compatibility, saving costs, and making operation easy; 7. High universality of imaging conditions: This invention amplifies the signal through a rolling circle amplification fluorescence in situ hybridization method, resulting in clear and bright signal points. No super-resolution imaging system is required; the signal can be interpreted using a confocal imaging system, and single-cell copy counting is also supported.

[0027] 8. This invention can detect single-base mutations in viral genes. The probe's RNA-binding arms A and B are complementary to the target sequence, forming an unclosed gap. SplintR ligase recognizes the precisely complementary bases at the 5' and 3' ends of the gap, thus ligating the gap to form a complete closed structure. A point mutation at either end will prevent ligation. This will allow for wider applications, such as in tumor mutation detection. 9. This invention can detect defective interfering RNA (DI RNA) generated during viral replication. DI RNA is subviral RNA produced by RNA viruses under the action of error-prone replicases, and is generally composed of discontinuous fragments on the genome. A probe set is designed targeting the boundaries of these discontinuous fragments. RNA-binding arms A and B identify the upstream sequence immediately adjacent to the boundary site, while the RNA target complementary region identifies the downstream sequence immediately adjacent to the boundary site. Through subsequent ligation-amplification-visualization, the spatial location information of the DI RNA can be identified.

[0028] In summary, this invention proposes a highly specific fluorescence in situ hybridization technique based on three asymmetric thermodynamic structural probes, two rounds of hybridization, and two ligation reactions. For the first time, it distinguishes and visualizes the vRNA, cRNA, and mRNA of influenza virus in situ at the single-cell scale, and can be extended to the detection of the three types of RNA of other negative-strand RNA viruses. Attached Figure Description

[0029] Figure 1 A schematic diagram comparing viral vRNA, cRNA, and mRNA; Figure 2 The probe design diagram for TriLock-FISH; Figure 3Flowchart for TriLock-FISH detection of viral vRNA, cRNA, and mRNA; Figure 4 The image shows the results of T TriLock-FISH in H1N1 influenza virus-infected cells, including vRNA, cRNA, and mRNA of all eight gene segments, as well as the results of RNA quantification analysis in a single cell. Figure 5 The image shows the effect of TriLock-FISH detection of vRNA, cRNA and mRNA of viral nucleoprotein genes in the lung tissue of mice infected with H1N1 influenza virus. Figure 6 The image shows the effect of TriLock-FISH detection of vRNA, cRNA, and mRNA of the nucleoprotein gene encoded by the plasmid in cell samples transfected with plasmids expressing H1N1 influenza virus RNA. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0031] This invention provides a method for in situ detection of negative-strand RNA viruses and differentiation of viral vRNA, cRNA, and mRNA, comprising the following steps: (1) The 5′ ends of probe P2 and probe P3 were phosphorylated using T4 polynucleotide kinase, respectively; probe P1 and phosphorylated probe P2 were incubated and then annealed to form probe P1 / P2 complex. (2) The samples to be tested were sequentially fixed, dehydrated, and permeabilized; (3) Add the prepared probe P1 / P2 complex to the pretreated sample and incubate it to allow probe P1 to pair with the RNA type site on the target RNA and guide probe P2 to the corresponding target site; wash away non-specifically bound probe P2. (4) Add phosphorylated probe P3 to align and pair it with the segment-specific internal sequence on the target RNA and the probe P1 / P2 complex. Probe P2 and probe P3 form two ligation gaps that are aligned end to end. Wash away non-specifically bound probe P3. (5) Using SplintR ligase and T4 DNA ligase, probe P2 and probe P3 are joined at the two gaps, forming a single-stranded closed circular DNA. (6) Using probe P1 as a primer and the single-stranded closed circular DNA formed in step (5) as a template, rolling circle amplification was performed using Phi29 DNA polymerase to generate amplification products. (7) Based on the fluorescent detection probe binding sequence of probe P3, a fluorescent detection probe fp is designed. The fluorescent detection probe fp is hybridized with the fluorescent detection probe binding sequence pre-placed by probe P3 on the amplification product to obtain a visible fluorescent signal. Using a confocal microscopy system for imaging, different types of RNA exhibit different fluorescent signals, thus enabling in situ detection and differentiation of three types of viral RNA.

[0032] Example 1: Based on the above FISH method, vRNA, cRNA, and mRNA were detected, differentiated, and quantified in H1N1 influenza virus-infected cells. 1. Probe Design Combination Figures 1-3 As shown, probes P1, P2, and P3 were designed for eight segments (HA, M, NA, NP, NS, PA, PB1, and PB2) of the negative-sense RNA virus of H1N1 influenza virus. The NCBI accession numbers for the eight cRNA segments are: PB2: FJ966079.1, PB1: FJ966080.1, PA: FJ966081.1, HA: FJ966082.1, NP: FJ966083.1, NA: FJ966084.1, M: FJ966085.1, and NS: FJ966086.1. The gene sequences of the eight segments of vRNA and the eight segments of cRNA are inversely complementary. The nucleotide sequences of the eight segments of mRNA, HA, M, NA, NP, NS, PA, PB1, and PB2, are shown in SEQ ID NO: 109 to SEQ ID NO: 116, respectively.

[0033] The probes P1 designed for the eight segments of vRNA are: P1-vRNA-HA, P1-vRNA-M, P1-vRNA-NA, P1-vRNA-NP, P1-vRNA-NS, P1-vRNA-PA, P1-vRNA-PB1, and P1-vRNA-PB2; the probes P2 designed for the eight segments of vRNA are: P2-vRNA-HA, P2-vRNA-M, P2-vRNA-NA, P2-vRNA-NP, P2-vRNA-NS, P2-vRNA-PA, P2-vRNA-PB1, and P2-vRNA-PB2; and the probes P3 designed for the eight segments of vRNA are: P3-vRNA-HA, P3-vRNA-M, P3-vRNA-NA, P3-vRNA-NP, P3-vRNA-NS, P3-vRNA-PA, P3-vRNA-PB1, and P3-vRNA-PB2.

[0034] The probes P1 designed for the eight segments of cRNA are: P1-cRNA-HA, P1-cRNA-M, P1-cRNA-NA, P1-cRNA-NP, P1-cRNA-NS, P1-cRNA-PA, P1-cRNA-PB1, and P1-cRNA-PB2; the probes P2 designed for the eight segments of cRNA are: P2-cRNA-HA, P2-cRNA-M, P2-cRNA-NA, P2-cRNA-NP, P2-cRNA-NS, P2-cRNA-PA, P2-cRNA-PB1, and P2-cRNA-PB2; and the probes P3 designed for the eight segments of cRNA are: P3-cRNA-HA, P3-cRNA-M, P3-cRNA-NA, P3-cRNA-NP, P3-cRNA-NS, P3-cRNA-PA, P3-cRNA-PB1, and P3-cRNA-PB2.

[0035] The probes P1 designed for the eight segments of mRNA are: P1-mRNA-HA, P1-mRNA-M, P1-mRNA-NA, P1-mRNA-NP, P1-mRNA-NS, P1-mRNA-PA, P1-mRNA-PB1, and P1-mRNA-PB2; the probes P2 designed for the eight segments of mRNA are: P2-mRNA-HA, P2-mRNA-M, P2-mRNA-NA, P2-mRNA-NP, P2-mRNA-NS, P2-mRNA-PA, P2-mRNA-PB1, and P2-mRNA-PB2; the probes P3 designed for the eight segments of mRNA are: P3-mRNA-HA, P3-mRNA-M, P3-mRNA-NA, P3-mRNA-NP, P3-mRNA-NS, P3-mRNA-PA, P3-mRNA-PB1, and P3-mRNA-PB2.

[0036] The specific probes are shown in Table 1.

[0037] Table 1 Probes of Example 1 2. Probe pretreatment (1) The 5' ends of all probes P2 and P3 were phosphorylated using T4 polynucleotide kinase.

[0038] (2) Mix all probe P1 and all phosphorylated probe P2 in a 1:1 ratio, incubate at high temperature (95°C, 2 minutes), and then slowly cool down to perform annealing treatment (cooling rate of 0.2°C per second, down to 25°C) to form probe P1 / P2 complex.

[0039] 3. Sample pretreatment This embodiment uses canine kidney epithelial cells (MDCK cells) for experiments. The cells are cultured to a sufficient quantity for the experiment, and this is done to facilitate subsequent operations.

[0040] (1) Culture the cells in glass-bottomed culture dishes until they reach 80% confluence, and then infect the cells with H1N1 influenza A virus for 8 hours (1 PFU / cell). Wash the cells three times with ice-cold PBST (PBS containing 0.1% Tween-20) to remove residual culture medium and other impurities.

[0041] (2) In order to maintain the cell morphology and keep the vRNA, cRNA and mRNA in their original positions in the cell, the cell sample was fixed with 4% paraformaldehyde (PFA), and after incubation for 24 h, the PFA was removed. The cells were washed three times with PBST at room temperature for 5 minutes each time to remove excess PFA.

[0042] (3) To remove the original water from the cells and facilitate the entry of reagents and reaction, pre-cooled 100% methanol was added to the fixed cells and immediately incubated at -80℃ for 15 minutes to dehydrate and denature the tissue. After the reaction was completed, the cell sample was taken out from -80℃ and placed at room temperature for equilibration for 5 minutes. Then the liquid was discarded, and the cells were washed three times with PBST at room temperature for 5 minutes each time.

[0043] (4) To increase cell membrane permeability and facilitate the entry of macromolecular reagents in subsequent reactions into the cells, 0.1 M hydrochloric acid solution was used to permeate the cell membrane, creating a porous structure. The cells were treated at room temperature for 5 minutes. After the reaction was completed, the liquid was discarded, and the cells were washed three times with PBST at room temperature for 5 minutes each time.

[0044] 4. First round of probe hybridization Add the hybridization system containing the probe P1 / P2 complex to the reaction chamber pretreated in step 3, place it in a humidified chamber, and hybridize overnight at 37°C (8–12 h). Wash cells three times with PBST for 10 min each time. The hybridization system containing the probe P1 / P2 complex consisted of: 10% formamide, 2×SSC, 0.1% Tween-20, 0.1 mg / mL salmon sperm DNA, and 500 nM probe P1 / P2 complex.

[0045] 5. Second round of probe hybridization The hybridization system containing all phosphorylated probe P3 was added to the reaction chamber after the first round of probe hybridization and placed in a humidified chamber for overnight hybridization at 37°C. The mixture was washed three times with PBST for 10 min each time. The hybridization system containing all phosphorylated probe P3 consisted of: 10% formamide, 2×SSC, 0.1% Tween-20, 0.1 mg / mL salmon sperm DNA, and 500 nM phosphorylated probe P3.

[0046] 6. Connection reaction After the probes hybridize to the target RNA and hybridize with each other, the gaps formed by vRNA, cRNA, and mRNA probes P2 and P3 need to be filled. At this point, the Splint R ligase reaction system and the T4 DNA ligase reaction system are mixed and added to the reaction chamber after the second round of probe hybridization. The mixture is incubated at 16°C for 1 hour, and then at 25°C for another hour. At this point, probes P2 and P3 are ligated into a complete single-stranded DNA loop. Cells are washed three times with PBST for 5 minutes each time. The Splint R ligase reaction system consists of 1 U / μL Splint R ligase and 1× ligase buffer; the T4 DNA ligase reaction system consists of 1 U / mL T4 DNA ligase and 1× ligase buffer.

[0047] 7. Rolling ring amplification reaction After the probe forms a circular shape, the captured information needs to be amplified to read the target sequence. This step uses rolling circle amplification (RoBAM) to amplify the lock-on probe. The RoBAM reaction mixture of phi29 DNA polymerase is mixed and added to the reaction chamber after the ligation reaction. The mixture is then incubated at 30°C for 2 hours. During this process, probe P1 acts as the primer for RCA initiation, using the DNA circle formed by probes P2 and P3 as a template for RoBAM amplification, thus amplifying the target signal. After the reaction, the liquid is discarded, and the cells are washed three times with PBST at room temperature for 5 minutes each time. The RoBAM reaction mixture of phi29 DNA polymerase consists of: 1 U / μL phi29 DNA polymerase, 1× buffer, 1 mM dNTPs, and 5% glycerol.

[0048] 8. Hybridization fluorescent detection probe Based on the fluorescent detection probe binding sequence in probe P3, fluorescent detection probes fp were designed as follows: Specifically, the fluorescent detection probes fp designed for the eight segments of vRNA are: fp-vRNA-HA, fp-vRNA-M, fp-vRNA-NA, fp-vRNA-NP, fp-vRNA-NS, fp-vRNA-PA, fp-vRNA-PB1, and fp-vRNA-PB2; the fluorescent detection probes fp designed for the eight segments of cRNA are: fp-cRNA-HA, fp- The fluorescent detection probes fp designed for eight segments of mRNA are: fp-mRNA-M, fp-cRNA-NA, fp-cRNA-NP, fp-cRNA-NS, fp-cRNA-PA, fp-cRNA-PB1, and fp-cRNA-PB2; the specific fluorescent detection probes fp designed for eight segments of mRNA are: fp-mRNA-HA, fp-mRNA-M, fp-mRNA-NA, fp-mRNA-NP, fp-mRNA-NS, fp-mRNA-PA, fp-mRNA-PB1, and fp-mRNA-PB2.

[0049] The specific fluorescent detection probes are shown in Table 2.

[0050] Table 2 Fluorescent detection probes of Example 1 All fluorescent detection probes were prepared into a hybridization system (PBS solution: 500 nM fluorescent detection probe, 2×SSC), added to the reaction chamber after the rolling circle amplification reaction, and incubated at 25°C for 3 h. After the reaction, the liquid was discarded, and the cells were washed three times with PBST at room temperature for 5 minutes each time. Then, DAPI staining was performed. A DAPI staining solution with a concentration of 0.1 μg / mL was prepared with PBS and added to the reaction chamber, and the reaction was carried out at room temperature for 2 min. Imaging was performed using a Nikon AX laser confocal microscope, with corresponding fluorescence channels set for different RNAs.

[0051] 9. Quantitative analysis of signals at the single-cell level The number of each RNA signal in the imaging was counted to obtain a single-cell level statistical result map.

[0052] The results are as follows Figure 4 As shown, the three types of influenza virus RNA can be clearly distinguished in each cell. Green represents the influenza virus vRNA, red represents the influenza virus cRNA, magenta represents the influenza virus mRNA, and blue represents the cell nucleus. cRNA is mainly distributed in the cell nucleus, while mRNA is mainly distributed in the cytoplasm, consistent with existing theories. Meanwhile, Figure 4 The statistical results show the average number of each RNA in a single infected cell, enabling quantitative analysis at the single-cell level.

[0053] Therefore, the method of the present invention can be applied to detect and distinguish negative-strand RNA virus vRNA, cRNA and mRNA in situ in virus-infected cells, and can achieve quantitative analysis of signals at the single-cell level.

[0054] Example 2: Based on the above FISH method, vRNA, cRNA, and mRNA encoded by nucleoprotein genes (NPs) were detected and distinguished in the lung tissue of mice infected with H1N1 influenza virus. 1. Probe Design Probes P1, P2, and P3 were designed to target the vRNA, cRNA, and mRNA of the nucleoprotein gene (NP, the same as the NP segment in Example 1) in the lung tissue of mice infected with H1N1 influenza virus.

[0055] The probes P1, P2, and P3 designed for NP targeting vRNA are specifically: P1-vRNA, P2-vRNA, and P3-vRNA; The probes P1, P2, and P3 designed for NP targeting cRNA are specifically: P1-cRNA, P2-cRNA, and P3-cRNA; The probes P1, P2, and P3 designed for NP targeting mRNA are specifically: P1-mRNA, P2-mRNA, and P3-mRNA.

[0056] The specific probes are shown in Table 3.

[0057] Table 3 Probes of Example 2 2. Probe pretreatment: Perform the same pretreatment steps as in Example 1.

[0058] 3. Sectioning and processing of lung tissue from mice infected with the virus (1) Babl / c mice aged 6-8 weeks were treated with 10 6 Intranasal inoculation with PFU for H1N1 influenza virus resulted in euthanasia after 3 days. Lung tissue was harvested, treated with 4% paraformaldehyde (PFA) for 24 h, embedded in OCT, and frozen to prepare 10 μM thick frozen sections. Before the experiment, the sections were fixed with 4% PFA for another 1 h, the PFA was discarded, and the sections were washed three times with PBST at room temperature for 5 minutes each time to remove excess PFA.

[0059] (2) To remove the original water from the tissue and facilitate the entry of reagents and reaction, pre-cooled 100% methanol was added to the fixed tissue, and it was immediately incubated at -80℃ for 15 minutes to dehydrate and denature the tissue. After the reaction was completed, the sample was taken out from -80℃, placed at room temperature for equilibration for 5 minutes, and then the liquid was discarded. The sample was washed three times with PBST at room temperature for 5 minutes each time.

[0060] (3) To increase cell permeability and facilitate the entry of macromolecular reagents in subsequent reactions into the cells, pepsin (2 mg / mL) was used to treat the cells for 10 min, followed by permeabilization of the cell membrane with 0.1 M hydrochloric acid solution for 5 min to create pores in the cell membrane. All reactions were carried out at 37°C. After the reaction was completed, the liquid was discarded, and the cells were washed three times with PBST at room temperature for 5 min each time.

[0061] 4. First round of probe hybridization Add the hybridization system containing the probe P1 / P2 complex to the reaction chamber pretreated in step 3, place it in a humidified chamber, and hybridize overnight at 37°C (8-12 h). Wash the cells three times with PBST for 10 min each time.

[0062] 5. Second round of probe hybridization: Same as step 5 in Example 1.

[0063] 6. Connection reaction: Same as step 6 in Example 1.

[0064] 7. Rolling ring amplification reaction: Same as step 7 in Example 1.

[0065] 8. Hybridization fluorescent detection probe Based on the fluorescent detection probe binding sequence of probe P3, fluorescent detection probes fp were designed as follows: fluorescent detection probe fp-vRNA designed for probe P3-vRNA; fluorescent detection probe fp-cRNA designed for probe P3-cRNA; and fluorescent detection probe fp-mRNA designed for probe P3-mRNA.

[0066] The specific fluorescent detection probes are shown in Table 4.

[0067] Table 4 Fluorescent detection probes of Example 2 Same as step 8 in Example 1.

[0068] like Figure 5 As shown, the three RNAs encoded by the influenza virus hemagglutinin gene can be clearly distinguished in infected lung tissue sections. Green represents the influenza virus vRNA, red represents the influenza virus cRNA, magenta represents the influenza virus mRNA, and blue represents the cell nucleus.

[0069] Therefore, the method of the present invention can be used to detect and distinguish vRNA, cRNA and mRNA of negative-strand RNA viruses in situ in tissue sections infected with viruses.

[0070] Example 3: Based on the above FISH method, the vRNA, cRNA, and mRNA of the NP gene derived from the plasmid were detected in cells transfected with a plasmid expressing the H1N1 influenza virus nucleoprotein gene (NP). 1. Probe Design Probes P1, P2, and P3 were designed to target the vRNA, cRNA, and mRNA of the plasmid-derived nucleoprotein gene (NP, the same as the NP segment in Example 1) in plasmid cells infected with H1N1 influenza virus.

[0071] The probes P1, P2, and P3 in this embodiment are the same as those in Embodiment 2.

[0072] 2. Probe pretreatment: Perform the same pretreatment steps as in Example 1.

[0073] 3. Sample pretreatment (1) The NP gene of influenza A (A / Puerto Rico / 8 / 1934) was reverse transcribed to obtain cDNA, which was then amplified by PCR to obtain a double-stranded DNA product. The double-stranded DNA product was treated with restriction endonucleases, ligated into the pHW2000 vector, and transformed into DH5α Escherichia coli. After colony PCR verification, the correct sequence of the cloned strain was obtained as a pHW2000-NP plasmid, which was used for subsequent viral recombination or protein expression.

[0074] This method is performed on cell samples. To culture a sufficient number of 293T cells for the experiment, and for ease of subsequent operations, the cells are first cultured in glass-bottomed culture dishes until 80% confluence, and then transfected with 500 ng of pHW2000-NP plasmid for 24 hours. The cells are then washed three times with pre-chilled PBST (PBS containing 0.1% Tween-20) on ice to remove residual culture medium and other impurities.

[0075] (2) In order to maintain the shape of the cells and keep the RNA in its original spatial position, the sample was fixed with 4% paraformaldehyde (PFA), and after incubation for 24 h, the PFA was removed. The sample was washed three times with PBST at room temperature for 5 minutes each time to remove excess PFA.

[0076] (3) To remove the original water from the cells and facilitate the entry of reagents and reaction, pre-cooled 100% methanol was added to the fixed cells and immediately incubated at -80℃ for 15 minutes to dehydrate and denature the tissue. After the reaction was completed, the sample was taken out from -80℃, placed at room temperature for equilibration for 5 minutes, and then the liquid was discarded. The sample was washed three times with PBST at room temperature for 5 minutes each time.

[0077] (4) To increase cell permeability and facilitate the entry of macromolecular reagents in subsequent reactions into the cells, 0.1M hydrochloric acid solution was used to permeate the cell membrane, creating a porous structure on the cell membrane. The cells were treated at room temperature for 5 minutes. After the reaction was completed, the liquid was discarded, and the cells were washed three times with PBST at room temperature for 5 minutes each time.

[0078] 4. First round of probe hybridization: Same as step 4 in Example 1.

[0079] 5. Second round of probe hybridization: Same as step 5 in Example 1.

[0080] 6. Connection reaction: Same as step 6 in Example 1.

[0081] 7. Rolling ring amplification reaction: Same as step 7 in Example 1.

[0082] 8. Hybridization fluorescent detection probe: The fluorescent detection probe in this embodiment is the same as in Embodiment 2. Follow step 8 of Embodiment 1.

[0083] The results are as follows Figure 6 As shown, the three types of RNA produced by the plasmid can be clearly distinguished in each cell. Green represents the influenza virus vRNA, red represents the influenza virus cRNA, magenta represents the influenza virus mRNA, and blue represents the cell nucleus.

[0084] Therefore, the method of the present invention can be used to detect and distinguish plasmid-derived vRNA, cRNA and mRNA in situ in plasmid-transfected cell samples.

[0085] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A probe array for in situ detection of negative-strand RNA viruses and differentiation of viral vRNA, cRNA, and mRNA, characterized in that: The probe combination includes any one or more combinations of probe combination 1 to probe combination 9; The probe combination 1 includes probes P1-vRNA-HA, P2-vRNA-HA, and P3-vRNA-HA for detecting viral vRNA, probes P1-cRNA-HA, P2-cRNA-HA, and P3-cRNA-HA for detecting cRNA, and probes P1-mRNA-HA, P2-mRNA-HA, and P3-mRNA-HA for detecting mRNA. The probe combination 2 includes probes P1-vRNA-M, P2-vRNA-M, and P3-vRNA-M for detecting viral vRNA, probes P1-cRNA-M, P2-cRNA-M, and P3-cRNA-M for detecting cRNA, and probes P1-mRNA-M, P2-mRNA-M, and P3-mRNA-M for detecting mRNA. The probe combination 3 includes probes P1-vRNA-NA, P2-vRNA-NA, and P3-vRNA-NA for detecting viral vRNA, probes P1-cRNA-NA, P2-cRNA-NA, and P3-cRNA-NA for detecting cRNA, and probes P1-mRNA-NA, P2-mRNA-NA, and P3-mRNA-NA for detecting mRNA. The probe combination 4 includes probes P1-vRNA-NP, P2-vRNA-NP, and P3-vRNA-NP for detecting viral vRNA, probes P1-cRNA-NP, P2-cRNA-NP, and P3-cRNA-NP for detecting cRNA, and probes P1-mRNA-NP, P2-mRNA-NP, and P3-mRNA-NP for detecting mRNA. The probe combination 5 includes probes P1-vRNA-NS, P2-vRNA-NS, and P3-vRNA-NS for detecting viral vRNA, probes P1-cRNA-NS, P2-cRNA-NS, and P3-cRNA-NS for detecting cRNA, and probes P1-mRNA-NS, P2-mRNA-NS, and P3-mRNA-NS for detecting mRNA. The probe combination 6 includes probes P1-vRNA-PA, P2-vRNA-PA, and P3-vRNA-PA for detecting viral vRNA, probes P1-cRNA-PA, P2-cRNA-PA, and P3-cRNA-PA for detecting cRNA, and probes P1-mRNA-PA, P2-mRNA-PA, and P3-mRNA-PA for detecting mRNA. The probe combination 7 includes probes P1-vRNA-PB1, P2-vRNA-PB1, and P3-vRNA-PB1 for detecting viral vRNA, probes P1-cRNA-PB1, P2-cRNA-PB1, and P3-cRNA-PB1 for detecting cRNA, and probes P1-mRNA-PB1, P2-mRNA-PB1, and P3-mRNA-PB1 for detecting mRNA. The probe combination 8 includes probes P1-vRNA-PB2, P2-vRNA-PB2, and P3-vRNA-PB2 for detecting viral vRNA, probes P1-cRNA-PB2, P2-cRNA-PB2, and P3-cRNA-PB2 for detecting cRNA, and probes P1-mRNA-PB2, P2-mRNA-PB2, and P3-mRNA-PB2 for detecting mRNA. The probe combination 9 includes probes P1-vRNA, P2-vRNA, and P3-vRNA for detecting viral vRNA, probes P1-cRNA, P2-cRNA, and P3-cRNA for detecting cRNA, and probes P1-mRNA, P2-mRNA, and P3-mRNA for detecting mRNA. The probe P1 is a viral RNA type identification probe and also serves as a primer for rolling circle amplification. Probe P1 contains an RNA target complementary region, a probe P2 complementary region, and a probe P3 complementary region. The target site of the RNA target complementary region is any one of the internal sequence of vRNA, the conserved 3′ end sequence of cRNA, and the poly(A) tail region of mRNA immediately adjacent to the coding region. The probe P2 is an RNA sequence origin recognition probe, comprising an RNA binding arm A and a P1 complementary arm A; the RNA binding arm A is complementary to the adjacent internal sequence of the target site of the RNA target complementary region on the target RNA, and the P1 complementary arm A is complementary to the P2 complementary region of the probe P1; the RNA binding arm A on the probe P2 is short while the P1 complementary arm A is long. The probe P3 is an RNA sequence source recognition probe, comprising an RNA binding arm B, a fluorescent detection probe binding sequence, and a P1 complementary arm B; the RNA binding arm B is complementary to the adjacent internal sequence of the complementary sequence of the RNA binding arm A on the target RNA, and the P1 complementary arm B is complementary to the P3 complementary region of the probe P1; the RNA binding arm B on the probe P3 is long while the P1 complementary arm B is short. When probes P1, P2, and P3 work synergistically on the same target RNA, probes P2 and P3, guided by probe P1, form two ligation gaps aligned end-to-end on the target RNA. After ligation, a single-stranded closed circular DNA is formed. Using probe P1 as a primer, rolling circle amplification is performed, generating a detectable fluorescent signal. The probes P1-vRNA-HA, P1-vRNA-M, P1-vRNA-NA, P1-vRNA-NP, P1-vRNA-NS, P1-vRNA-PA, P1-vRNA-PB1, P1-vRNA-PB2, P1-cRNA-HA, P1-cRNA-M, P1-cRNA-NA, P1-cRNA-NP, P1-cRNA-NS, P1-cRN The nucleotide sequences of A-PA, P1-cRNA-PB1, P1-cRNA-PB2, P1-mRNA-HA, P1-mRNA-M, P1-mRNA-NA, P1-mRNA-NP, P1-mRNA-NS, P1-mRNA-PA, P1-mRNA-PB1, P1-mRNA-PB2, P1-vRNA, P1-cRNA, and P1-mRNA are as shown in SEQ. ID NO: 1~SEQ ID NO: 27; The nucleotide sequences of the probes P2-vRNA-HA, P2-vRNA-M, P2-vRNA-NA, P2-vRNA-NP, P2-vRNA-NS, P2-vRNA-PA, P2-vRNA-PB1, P2-vRNA-PB2, P2-cRNA-HA, P2-cRNA-M, P2-cRNA-NA, P2-cRNA-NP, P2-cRNA-NS, P2-cRNA-PA, P2-cRNA-PB1, P2-cRNA-PB2, P2-mRNA-HA, P2-mRNA-M, P2-mRNA-NA, P2-mRNA-NP, P2-mRNA-NS, P2-mRNA-PA, P2-mRNA-PB1, P2-mRNA-PB2, P2-vRNA, P2-cRNA, and P2-mRNA are shown in SEQ ID NO: 28 to SEQ ID NO: 54, respectively. The probes P3-vRNA-HA, P3-vRNA-M, P3-vRNA-NA, P3-vRNA-NP, P3-vRNA-NS, P3-vRNA-PA, P3-vRNA-PB1, P3-vRNA-PB2, P3-cRNA-HA, P3-cRNA-M, P3-cRNA-NA, P3-cRNA-NP, P3-cRNA-NS, P3-cRN The nucleotide sequences of A-PA, P3-cRNA-PB1, P3-cRNA-PB2, P3-mRNA-HA, P3-mRNA-M, P3-mRNA-NA, P3-mRNA-NP, P3-mRNA-NS, P3-mRNA-PA, P3-mRNA-PB1, P3-mRNA-PB2, P3-vRNA, P3-cRNA, and P3-mRNA are as shown in SEQ. ID NO: 55~SEQ ID NO: 81; The negative-strand RNA virus is influenza A virus H1N1; When probe P3 is P3-vRNA-HA, the corresponding fluorescent detection probe fp is fp-vRNA-HA; when probe P3 is P3-vRNA-M, the corresponding fluorescent detection probe fp is fp-vRNA-M; when probe P3 is P3-vRNA-NA, the corresponding fluorescent detection probe fp is fp-vRNA-NA; when probe P3 is P3-vRNA-NP, the corresponding fluorescent detection probe fp is fp-vRNA-NP; when probe P3 is P3-vRNA-NS, the corresponding fluorescent detection probe fp is fp-vRNA-NS; when probe P3 is P3-vRNA-PA, the corresponding fluorescent detection probe fp is fp- vRNA-PA; when probe P3 is P3-vRNA-PB1, the corresponding fluorescent detection probe fp is fp-vRNA-PB1; when probe P3 is P3-vRNA-PB2, the corresponding fluorescent detection probe fp is fp-vRNA-PB2; when probe P3 is P3-cRNA-HA, the corresponding fluorescent detection probe fp is fp-cRNA-HA; when probe P3 is P3-cRNA-M, the corresponding fluorescent detection probe fp is fp-cRNA-M; when probe P3 is P3-cRNA-NA, the corresponding fluorescent detection probe fp is fp-cRNA-NA; when probe P3 is P3-cRNA-NP, the corresponding fluorescent... When the optical detection probe fp is fp-cRNA-NP; when probe P3 is P3-cRNA-NS, the corresponding fluorescent detection probe fp is fp-cRNA-NS; when probe P3 is P3-cRNA-PA, the corresponding fluorescent detection probe fp is fp-cRNA-PA; when probe P3 is P3-cRNA-PB1, the corresponding fluorescent detection probe fp is fp-cRNA-PB1; when probe P3 is P3-cRNA-PB2, the corresponding fluorescent detection probe fp is fp-cRNA-PB2; when probe P3 is P3-mRNA-HA, the corresponding fluorescent detection probe fp is fp-mRNA-HA; when probe P3 is P3... When P3 is P3-mRNA-M, the corresponding fluorescent detection probe fp is fp-mRNA-M; when P3 is P3-mRNA-NA, the corresponding fluorescent detection probe fp is fp-mRNA-NA; when P3 is P3-mRNA-NP, the corresponding fluorescent detection probe fp is fp-mRNA-NP; when P3 is P3-mRNA-NS, the corresponding fluorescent detection probe fp is fp-mRNA-NS; when P3 is P3-mRNA-PA, the corresponding fluorescent detection probe fp is fp-mRNA-PA; when P3 is P3-mRNA-PB1, the corresponding fluorescent detection probe fp is fp-mRNA-PB1.When probe P3 is P3-mRNA-PB2, the corresponding fluorescent detection probe fp is fp-mRNA-PB2; when probe P3 is P3-vRNA, the corresponding fluorescent detection probe fp is fp-vRNA; when probe P3 is P3-cRNA, the corresponding fluorescent detection probe fp is fp-cRNA; when probe P3 is P3-mRNA, the corresponding fluorescent detection probe fp is fp-mRNA. The nucleotide sequences of the fluorescent detection probes fp-vRNA-HA, fp-vRNA-M, fp-vRNA-NA, fp-vRNA-NP, fp-vRNA-NS, fp-vRNA-PA, fp-vRNA-PB1, fp-vRNA-PB2, fp-cRNA-HA, fp-cRNA-M, fp-cRNA-NA, fp-cRNA-NP, fp-cRNA-NS, fp-cRNA-PA, fp-cRNA-PB1, fp-cRNA-PB2, fp-mRNA-HA, fp-mRNA-M, fp-mRNA-NA, fp-mRNA-NP, fp-mRNA-NS, fp-mRNA-PA, fp-mRNA-PB1, fp-mRNA-PB2, fp-vRNA, fp-cRNA, and fp-mRNA are shown in SEQ ID NO: 82 to SEQ ID NO: 108, respectively.

2. A kit for in situ detection of negative-strand RNA viruses and differentiation of viral vRNA, cRNA, and mRNA, characterized in that: The kit comprises the probe combination as described in claim 1.

Images