A multiplex qPCR primer probe set, kit, detection method and application for detecting alphaviruses

Abstract

The application discloses a multiplex qPCR primer probe set, a kit, a detection method and application for detecting alphaviruses, and belongs to the technical field of molecular biological detection. The application designs a set of primer probe combination for a plurality of alphaviruses capable of infecting insect cell lines, including western equine encephalitis virus, eastern equine encephalitis virus, sindbis virus, chikungunya virus, AURA virus and Venezuelan equine encephalitis virus. The combination realizes synchronous detection and identification of a plurality of target viruses in one reaction system. The application also provides a kit containing the primer probe set and a corresponding detection method. The detection method has the advantages of wide coverage, high detection sensitivity, strong specificity, good anti-matrix interference capability and the like, and is suitable for safety detection and quality control of alphavirus contamination in insect cell banks, cell cultures and related biological products.

Description

Technical Field

[0001] This invention belongs to the field of molecular biological detection technology, and particularly relates to a multiplex qPCR primer and probe set, kit, detection method and application for detecting alphavirus. Background Technology

[0002] Alphaviruses belong to the Togaviridae family and are a type of enveloped, single-stranded, positive-sense RNA virus that can be transmitted via mosquitoes, making them typical arboviruses.

[0003] Most alpha viruses are zoonotic. Among them, Venezuelan equine encephalitis virus (VEEV) is highly infectious and pathogenic to humans. It can be transmitted through horses and is easily transmitted to humans via aerosols, causing fetal malformations. It has become an important target for laboratory and public health safety. Eastern equine encephalitis virus (EEEV) is also one of the most deadly mosquito-borne viruses, with extremely strong neuropathogenicity. Western equine encephalitis virus (WEEV) is more susceptible to infants and the elderly with weakened immune systems. Even after recovery, severely ill patients still have the risk of teratogenic sequelae. Avira not only threaten the human nervous system but also cause severe damage to joints: infection with Chikungunya Virus (CHIKV) leads to intense and persistent joint pain, significantly impacting daily life; Sindbis Virus (SINV), while less pathogenic, is a well-established human pathogen as the prototype and model species of the alphavirus genus and is widely used as a tool for expressing exogenous genes in vaccine development and gene therapy; Aura Virus (AURAV) is a potential anti-cancer tool due to its unique genetic structure—it can specifically replicate in tumor cells and has an extremely low infection rate in humans—and is used in research on oncolytic virus therapy. To ensure the safety of biological products, regulatory agencies such as the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), the U.S. Food and Drug Administration (FDA), and the Pharmacopoeia of the People's Republic of China all explicitly require the detection and risk assessment of exogenous viral factors in cell substrates used in production. Therefore, there is a widespread demand for alpha virus detection in areas such as environmental monitoring, epidemic prevention and control, and quality control of biological products.

[0004] Currently, traditional methods for detecting alpha 1 virus mainly include serological testing, cytopathic effect (CPE) assays, electron microscopy, and nucleic acid detection. However, these methods have significant limitations: the specificity and sensitivity of serological testing are easily affected by antibody quality, and alpha 1 virus antibodies may cross-react, interfering with diagnostic results; cell culture methods are time-consuming (usually requiring several weeks), have limited sensitivity, and carry high operational risks; electron microscopy is expensive and requires specialized operation; while real-time quantitative PCR (qPCR) technology, due to its high sensitivity, high specificity, quantitative capability, and rapid efficiency, has become the gold standard for pathogen detection.

[0005] However, current technologies still lack a systematically validated multiplex qPCR detection protocol that can simultaneously cover multiple key alphaviruses and is optimized for insect cells and their product matrices. Most existing primer-probe combinations target only a single or a few alphaviruses, resulting in cumbersome detection procedures, increased costs, and an inability to identify and detect multiple target viruses in a single reaction, thus failing to fully meet the urgent need for comprehensive viral safety assessment.

[0006] Therefore, there is an urgent need in this field to develop a primer-probe combination and supporting method for multiplex qPCR detection that has broad coverage, high sensitivity, strong specificity, good resistance to matrix interference, and is suitable for the quality control of insect cells and their biological products. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a multiplex qPCR primer-probe set for detecting alphaviruses, which can detect a variety of alphaviruses that can infect insect cells, and has high detection sensitivity, good specificity and strong durability.

[0008] Another object of the present invention is to provide an application of the aforementioned multiplex qPCR primer-probe set in the preparation of products for detecting alphavirus.

[0009] Another object of the present invention is to provide a multiplex qPCR kit for detecting alphavirus.

[0010] Another object of the present invention is to provide a method for detecting various alphaviruses that can infect insect cells for non-diagnostic purposes.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a multiplex qPCR primer-probe set for detecting alphavirus, comprising an upstream primer WEEV-F, a downstream primer WEEV-R, and a fluorescent probe WEEV-P for detecting western equine encephalitis virus. The nucleotide sequence of the upstream primer WEEV-F is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer WEEV-R is shown in SEQ ID NO.2, and the nucleotide sequence of the fluorescent probe WEEV-P is shown in SEQ ID NO.3. The upstream primer EEEV-F, the downstream primer EEEV-R, and the fluorescent probe EEEV-P are used to detect eastern equine encephalitis virus. The nucleotide sequence of the upstream primer EEEV-F is shown in SEQ ID NO.4, the nucleotide sequence of the downstream primer EEEV-R is shown in SEQ ID NO.5, and the nucleotide sequence of the fluorescent probe EEEV-P is shown in SEQ ID NO.6. The upstream primer SINV-F, the downstream primer SINV-R, and the fluorescent probe SINV-P are used for detecting Sinderby virus. The nucleotide sequence of the upstream primer SINV-F is shown in SEQ ID NO.7, the nucleotide sequence of the downstream primer SINV-R is shown in SEQ ID NO.8, and the nucleotide sequence of the fluorescent probe SINV-P is shown in SEQ ID NO.9. The upstream primer CHIKV-F, the downstream primer CHIKV-R, and the fluorescent probe CHIKV-P for detecting Chikungunya virus are shown in SEQ ID NO.10, the nucleotide sequence of the upstream primer CHIKV-F is shown in SEQ ID NO.11, and the nucleotide sequence of the fluorescent probe CHIKV-P is shown in SEQ ID NO.12. The upstream primer AURAV-F, the downstream primer AURAV-R, and the fluorescent probe AURAV-P for detecting AURA virus are shown in SEQ ID NO.13, the nucleotide sequence of the upstream primer AURAV-F is shown in SEQ ID NO.14, and the nucleotide sequence of the fluorescent probe AURAV-P is shown in SEQ ID NO.15. The upstream primer VEEV-F, the downstream primer VEEV-R, and the fluorescent probe VEEV-P are used to detect Venezuelan equine encephalitis virus. The nucleotide sequence of the upstream primer VEEV-F is shown in SEQ ID NO.16, the nucleotide sequence of the downstream primer VEEV-R is shown in SEQ ID NO.17, and the nucleotide sequence of the fluorescent probe VEEV-P is shown in SEQ ID NO.18.

[0012] Preferably, the fluorescent probes in the primer-probe set are labeled with a fluorescent reporter group at their 5' end and a fluorescent quencher group at their 3' end.

[0013] Preferably, the fluorescent reporter group includes any one of FAM, VIC, ROX or CY5; and / or the fluorescent quencher group includes BHQ1 or BHQ2.

[0014] This invention also provides the application of the aforementioned multiplex qPCR primer-probe set in the preparation of products for detecting alphavirus.

[0015] The present invention also provides a multiplex qPCR kit for detecting alphavirus, comprising the multiplex qPCR primer and probe set.

[0016] Preferably, the detection system of the multiplex qPCR kit, based on a total volume of 25 μL, comprises the following components: 1 μL of nucleic acid-free water, 13 μL of qPCR reaction solution, 0.5 μL of primer mixing working solution, 0.5 μL of probe mixing working solution, and 10 μL of template.

[0017] Preferably, in the primer mixed working solution, the final concentrations of the upstream primer EEEV-F, downstream primer EEEV-R, upstream primer SINV-F, downstream primer SINV-R, upstream primer CHIKV-F, downstream primer CHIKV-R, upstream primer AURAV-F, downstream primer AURAV-R, upstream primer VEEV-F, and downstream primer VEEV-R are all 10 μM; and the final concentrations of the upstream primer WEEV-F and downstream primer WEEV-R in the primer mixed working solution are both 3 μM.

[0018] Preferably, in the probe mixed working solution, the final concentrations of fluorescent probes EEEV-P, SINV-P, CHIKV-P, AURAV-P, and VEEV-P are all 10 μM; and the final concentration of fluorescent probe WEEV-P is 3 μM.

[0019] This invention also provides a method for detecting various alphaviruses that can infect insect cells for non-diagnostic purposes, comprising the following steps: extracting RNA from the sample to be tested and reverse transcribing it into cDNA; using the cDNA as a template, performing qPCR amplification using the multiplex qPCR primer and probe set or the multiplex qPCR kit; collecting fluorescence signals, and determining whether the sample is positive for Western equine encephalitis virus, Eastern equine encephalitis virus, Sindbis virus, Chikungunya virus, AURA virus, and Venezuelan equine encephalitis virus based on the amplification curve and Cp value.

[0020] Preferably, the criteria for judgment are as follows: if the Cp value of the sample to be tested is less than 40 and there is an obvious amplification curve, it is judged as positive; if the Cp value of the sample to be tested is greater than or equal to 40 or there is no obvious amplification curve, it is judged as negative.

[0021] Preferably, the qPCR reaction program is as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 60℃ annealing for 1 min, 45 cycles; 40℃ cooling for 30 s.

[0022] The beneficial effects of this invention are: This invention provides a multiplex qPCR primer-probe set for detecting alphaviruses. This primer-probe set has broad coverage and can detect a variety of alphaviruses that can infect insect cells, namely Western equine encephalitis virus, Eastern equine encephalitis virus, Sindbis virus, Chikungunya virus, AURA virus, and Venezuelan equine encephalitis virus. It exhibits high detection sensitivity, with 100% detection of plasmid standards at 10 copies / reaction for each alphavirus. The results show good specificity and robustness, and the nucleic acid extraction and detection processes are unaffected by cell matrix interference. Each alphavirus plasmid standard shows a positive S-shaped amplification curve in its corresponding qPCR reaction system, enabling precise molecular diagnosis of various alphaviruses. It is suitable for quality control of insect cells and their biological products and has significant application value in pathogen detection and epidemiological investigations. Attached Figure Description

[0023] Figure 1 This is the standard curve for qPCR fluorescence quantitative detection of western equine encephalitis virus in Example 2.

[0024] Figure 2 This is the standard curve for qPCR fluorescence quantitative detection of Eastern Equine Encephalitis Virus in Example 2.

[0025] Figure 3 This is the standard curve for qPCR fluorescence quantitative detection of Sinderby virus in Example 2.

[0026] Figure 4 This is the standard curve for qPCR fluorescence quantitative detection of chikungunya virus in Example 2.

[0027] Figure 5 This is the standard curve for qPCR fluorescence quantitative detection of AURA virus in Example 2.

[0028] Figure 6 This is the standard curve for qPCR fluorescence quantitative detection of Venezuelan equine encephalitis virus in Example 2.

[0029] Figure 7 The detection curve for qPCR fluorescence quantitative detection of western equine encephalitis virus in Example 2 is shown.

[0030] Figure 8 The detection curve for qPCR fluorescence quantitative detection of Eastern Equine Encephalitis Virus in Example 2 is shown.

[0031] Figure 9 This is the detection curve for qPCR fluorescence quantitative detection of Sinderby virus in Example 2.

[0032] Figure 10 This is the detection curve for qPCR fluorescence quantitative detection of chikungunya virus in Example 2.

[0033] Figure 11 The detection curve for qPCR fluorescence quantitative detection of AURA virus in Example 2 is shown.

[0034] Figure 12 This is the detection curve for qPCR fluorescence quantitative detection of Venezuelan equine encephalitis virus in Example 2.

[0035] Figure 13 The detection curve for the primer set optimized for Eastern Equine Encephalitis Virus in Example 6.

[0036] Figure 14 The detection curve for western equine encephalitis virus before primer and probe concentration optimization in Example 7 is shown.

[0037] Figure 15 The detection curve for western equine encephalitis virus after optimization of primer and probe concentrations in Example 7 is shown. Detailed Implementation

[0038] This invention provides a multiplex qPCR primer-probe set for detecting alphavirus, comprising an upstream primer WEEV-F, a downstream primer WEEV-R, and a fluorescent probe WEEV-P for detecting western equine encephalitis virus. The nucleotide sequence of the upstream primer WEEV-F is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer WEEV-R is shown in SEQ ID NO.2, and the nucleotide sequence of the fluorescent probe WEEV-P is shown in SEQ ID NO.3. The upstream primer EEEV-F, the downstream primer EEEV-R, and the fluorescent probe EEEV-P are used to detect eastern equine encephalitis virus. The nucleotide sequence of the upstream primer EEEV-F is shown in SEQ ID NO.4, the nucleotide sequence of the downstream primer EEEV-R is shown in SEQ ID NO.5, and the nucleotide sequence of the fluorescent probe EEEV-P is shown in SEQ ID NO.6. The upstream primer SINV-F, the downstream primer SINV-R, and the fluorescent probe SINV-P are used for detecting Sinderby virus. The nucleotide sequence of the upstream primer SINV-F is shown in SEQ ID NO.7, the nucleotide sequence of the downstream primer SINV-R is shown in SEQ ID NO.8, and the nucleotide sequence of the fluorescent probe SINV-P is shown in SEQ ID NO.9. The upstream primer CHIKV-F, the downstream primer CHIKV-R, and the fluorescent probe CHIKV-P for detecting Chikungunya virus are shown in SEQ ID NO.10, the nucleotide sequence of the upstream primer CHIKV-F is shown in SEQ ID NO.11, and the nucleotide sequence of the fluorescent probe CHIKV-P is shown in SEQ ID NO.12. The upstream primer AURAV-F, the downstream primer AURAV-R, and the fluorescent probe AURAV-P for detecting AURA virus are shown in SEQ ID NO.13, the nucleotide sequence of the upstream primer AURAV-F is shown in SEQ ID NO.14, and the nucleotide sequence of the fluorescent probe AURAV-P is shown in SEQ ID NO.15. The upstream primer VEEV-F, the downstream primer VEEV-R, and the fluorescent probe VEEV-P are used to detect Venezuelan equine encephalitis virus. The nucleotide sequence of the upstream primer VEEV-F is shown in SEQ ID NO.16, the nucleotide sequence of the downstream primer VEEV-R is shown in SEQ ID NO.17, and the nucleotide sequence of the fluorescent probe VEEV-P is shown in SEQ ID NO.18.

[0039] In this invention, the fluorescent probes in the primer-probe set are labeled with a fluorescent reporter group at their 5' end and a fluorescent quencher group at their 3' end. Different fluorescent reporter groups are used to differentially label the probes corresponding to different viruses within the same reaction system. Different fluorescence channels are used to distinguish and detect multiple alphaviruses. The presence or type of alphavirus in the sample can be identified based on the fluorescence signal and amplification curve in the amplification product. This invention does not limit the specific fluorescent reporter and quencher groups contained in the probes; any group that can connect to the probe and play a corresponding characterizing role is acceptable. As one possible implementation, the fluorescent reporter group preferably includes any one of FAM, VIC, ROX, or CY5; the fluorescent quencher group preferably includes BHQ1 or BHQ2. In some embodiments, WEEV-P contains FAM, EEEV-P contains VIC, SINV-P contains ROX, CHIKV-P contains CY5, AURAV-P contains FAM, and VEEV-P contains VIC; in some embodiments, WEEV-P, EEEV-P, AURAV-P, and VEEV-P contain BHQ1, and SINV-P and CHIKV-P contain BHQ2.

[0040] This invention employs bioanalytical techniques to classify and compare all nucleic acid information of Western Equine Encephalitis Virus (WEEV), Eastern Equine Encephalitis Virus (EEEV), Sindbis Virus (SINV), Chikungunya Virus (CHIKV), Aura Virus (AURAV), and Venezuelan Equine Encephalitis Virus (VEEV) in Genebank. Conserved regions that maximally cover specific alphavirus species and their variants or subtypes are extracted for primer and probe design. Furthermore, addressing the abnormal amplification curve of Eastern Equine Encephalitis Virus (EEEV), primer sequences undergo multiple rounds of screening and structural optimization. Ultimately, primer-probe combinations for qPCR detection of each alphavirus are selected, ensuring stable and reliable detection results for multiple alphaviruses.

[0041] This invention also provides the application of the aforementioned multiplex qPCR primer-probe set in the preparation of products for detecting alphavirus.

[0042] The present invention also provides a multiplex qPCR kit for detecting alphavirus, comprising the multiplex qPCR primer and probe set.

[0043] In this invention, the kit preferably further includes at least one of a negative control, a positive control, and a qPCR reaction solution. The positive control is a plasmid standard for alpha virus, and its detection sequence is preferably shown in SEQ ID NO. 19-20. The qPCR reaction solution is a commonly used component for qPCR reactions, and the specific reaction reagents include a thermostable DNA polymerase for qPCR, buffer, dNTPs, and Mg2+. 2+ The present invention does not limit the use of reagents such as stabilizers. As an optional implementation, the qPCR reaction solution in the kit of the present invention can be pre-mixed and aliquoted with the primer-probe combination of the present invention. During use, only the reverse-transcribed genomic nucleic acid needs to be added to the premixed system for detection, reducing potential contamination during preparation and providing convenience and speed. By including positive and negative controls, the present invention effectively avoids false negative or false positive results, further ensuring the accuracy of the detection.

[0044] In some embodiments, the detection system of the multiplex qPCR kit preferably includes the following components in a total volume of 25 μL: 1 μL of nucleic acid-free water, 13 μL of qPCR reaction solution, 0.5 μL of primer mixing working solution, 0.5 μL of probe mixing working solution, and 10 μL of template.

[0045] In some embodiments, the qPCR reaction solution preferably includes LightCycler® 480 Probe Master.

[0046] In some embodiments, the final concentrations of the upstream primer EEEV-F, downstream primer EEEV-R, upstream primer SINV-F, downstream primer SINV-R, upstream primer CHIKV-F, downstream primer CHIKV-R, upstream primer AURAV-F, downstream primer AURAV-R, upstream primer VEEV-F, and downstream primer VEEV-R in the primer mixture are preferably all 10 μM; the final concentrations of the upstream primer WEEV-F and downstream primer WEEV-R in the primer mixture are preferably both 3 μM.

[0047] In some embodiments, the final concentrations of fluorescent probes EEEV-P, SINV-P, CHIKV-P, AURAV-P, and VEEV-P in the probe mixed working solution are preferably all 10 μM; the final concentration of fluorescent probe WEEV-P in the probe mixed working solution is preferably 3 μM.

[0048] To address the abnormal amplification curve of Western Equine Encephalitis Virus (WEEV) in qPCR reaction systems, this invention optimizes primer and probe concentrations, identifying the optimal primer and probe concentration combination suitable for the detection system. This effectively corrects the amplification curve morphology, ensuring a stable amplification trend and normal signal response during qPCR detection, thereby improving the stability and accuracy of the detection.

[0049] This invention also provides a method for detecting various alphaviruses that can infect insect cells for non-diagnostic purposes, comprising the following steps: extracting RNA from the sample to be tested and reverse transcribing it into cDNA; using the cDNA as a template, performing qPCR amplification using the multiplex qPCR primer and probe set or the multiplex qPCR kit; collecting fluorescence signals, and determining whether the sample is positive for Western equine encephalitis virus, Eastern equine encephalitis virus, Sindbis virus, Chikungunya virus, AURA virus, and Venezuelan equine encephalitis virus based on the amplification curve and Cp value. The method provided by this invention for detecting various alphaviruses that can infect insect cells is a method for non-disease diagnosis and treatment purposes, referring to the detection of alphavirus infection in non-insect animals, used for exogenous virus risk screening and sample contamination detection, including but not limited to the detection of alphavirus contamination in environments / scenarios such as air, soil, biological products, and laboratory samples, realizing the screening and identification of alphavirus contamination in different environments and application scenarios, fully meeting the actual detection needs in fields such as environmental monitoring, epidemic source tracing and prevention, biological product quality and safety control, and laboratory pathogen screening.

[0050] This invention does not impose specific limitations on the methods for extracting RNA from samples and performing reverse transcription. Conventional methods well-known in the art can be used to extract RNA from the samples and perform reverse transcription; alternatively, commercially available kits can be used. The detection method established by this invention can simultaneously detect and identify multiple target viruses in the same reaction system, or it can perform single-virus detection. As one possible implementation, this invention utilizes a quadruple qPCR detection method to detect Western equine encephalitis virus, Eastern equine encephalitis virus, Sindbis virus, and Chikungunya virus; as another possible implementation, this invention utilizes a dual qPCR detection method to detect AURA virus and Venezuelan equine encephalitis virus. This invention is not limited to the above-mentioned group detection methods. According to actual detection needs, the six alphaviruses can be freely combined and arbitrarily configured to flexibly construct qPCR reaction systems with different ratios. This allows for the selection of any single virus for individual detection, or the simultaneous detection of two or more viruses through arbitrary combinations, making it suitable for virus combination screening needs in different detection scenarios. The method of this invention has the advantages of being rapid, sensitive, and efficient.

[0051] In this invention, if multiple target viruses are simultaneously detected in the same reaction system, the template in the reaction system is a mixed template. Preferably, the templates of each target virus in the reaction system are mixed in an equal volume ratio. In some embodiments, when simultaneously detecting and identifying four viruses—Western equine encephalitis virus, Eastern equine encephalitis virus, Sindbis virus, and Chikungunya virus—1-2.5 μL of each pathogen template is thoroughly mixed, and the resulting mixed template is used as the mixed template.

[0052] In this invention, the preferred criteria for judgment are as follows: if the Cp value of the sample to be tested is less than 40 and there is an obvious amplification curve, it is judged as positive; if the Cp value of the sample to be tested is greater than or equal to 40 or there is no obvious amplification curve, it is judged as negative.

[0053] In this invention, the preferred qPCR reaction program is: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 60℃ annealing for 1 min, 45 cycles; and 40℃ cooling for 30 s. This invention optimizes the annealing temperature to ensure good stability of the amplification reaction.

[0054] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0055] Unless otherwise specified, the following embodiments are all conventional methods.

[0056] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0057] Example 1: Design of Primer-Probe Combinations This invention summarizes a variety of alphaviruses that can infect insect cells and designs primer-probe combinations for qPCR detection of these viruses. Information on these alphaviruses that can infect insect cells is shown in Table 1 below.

[0058] Table 1. Types of alphaviruses that can infect insect cells.

[0059] This invention employs bioanalytical techniques to classify and compare all nucleic acid information of the alphavirus species shown in Table 1 in Genebank, extracting conserved regions that maximally cover specific alphavirus species and their variants or subtypes for primer and probe design. Finally, primer-probe combinations for qPCR detection against each alphavirus were selected, as shown in Table 2 below.

[0060] Table 2 Primer-probe combinations for alphaviruses that can infect insect cells. Example 2: Detection of the linear range of alphaviruses capable of infecting insect cells using primer-probe combinations The corresponding plasmid standards for each virus were prepared from 1×10⁻⁶. 7 The concentration was sequentially diluted 10-fold in 7 gradients, each with a concentration of 1×10⁻⁶ copies / μL. 7 1×10 6 1×10 5 1×10 4 1×10 3 1×10 2 and 1×10 1 Copy / μL, then take 10μL for qPCR reaction and plot a standard curve.

[0061] The plasmid standards were pUC57-WEEV-EEEV-SINV-CHIKV and pUC57-AURAV-VEEV, respectively. The target gene sequences of the plasmid standards are shown in Table 3 below.

[0062] Table 3. Target gene sequences for plasmid standards.

[0063] The premixed solutions for qPCR detection were prepared, and their composition is shown in Table 4 below. The total volume for each reaction was 25 μL, containing 15 μL of the premixed solution shown in the table below, and 10 μL of template (plasmid standard or sample cDNA). The primer and probe mixing working solutions for each system were prepared as shown in Table 5 below. The qPCR reaction program was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 60℃ annealing for 1 min (fluorescence was collected at this step), 45 cycles; cooling at 40℃ for 30 s. The fluorescence detection channel selected was FAM+VIC+ROX+CY5.

[0064] Table 4. Premixes for qPCR detection of insect cell alphavirus.

[0065] Table 5. Primer and probe mixtures for various systems capable of infecting insect cell alphavirus.

[0066] The corresponding standard plasmids for each virus are in 1×10 7 ~1×10 1 The standard curves for 7 gradient qPCR quantitative detection at copies / μL are shown below. Figures 1-6 As shown, the detection curve is as follows Figures 7-12 As shown, it can be seen that the corresponding standard plasmids for each virus are within 1×1027 ~1×10 1 All seven gradients at copy / μL were detectable, and the correlation coefficient R was [value missing]. 2 ≥0.99, with a slope between -3.1 and -3.58, indicating a good linear relationship.

[0067] Example 3: Detection Limit of Primer-Probe Combination for Infecting Insect Cell Alphavirus This embodiment evaluates the detection limit of a method for detecting insect-infectable cell alphaviruses. Using the qPCR system and method of Example 2, plasmid standards of 10 copies / reaction for each virus were detected; each concentration was tested repeatedly, with a total of 10 replicates per concentration. All concentrations must be detected as positive; simultaneously, the detection limit Cp value of each virus plasmid must meet a relative standard deviation (RSD) ≤ 5%, meaning that the sensitivity point can be detected repeatedly and stably.

[0068] The test results are shown in Tables 6-11. All plasmid standards with 10 copies / reaction of each alphavirus were detected 100%, and the RSD of the Cp values ​​were all ≤5%, indicating high detection sensitivity and good repeatability.

[0069] Table 6. Limit of Detection (LOD) and Cp Value of Plasmid Standards Corresponding to WEEV Virus A

[0070] Table 7. Limit of Detection (LOD) and Cp Value of Plasmid Standards Corresponding to EEEV Virus A

[0071] Table 8. Limit of Detection (LOD) and Cp Value of Plasmid Standards for SINV (SiV) A Virus

[0072] Table 9. Detection Limits and Cp Values ​​of Plasmid Standards Corresponding to CHIKV Virus

[0073] Table 10. Limit of Detection (LOD) and Cp Values ​​for Plasmid Standards Corresponding to AURAV Virus 10

[0074] Table 11 Detection Limits and Cp Values ​​of Plasmid Standards Corresponding to VEEV (Veinovirus A)

[0075] Example 4: Verification of the robustness of primer-probe combination detection for insect cell alphavirus infection This embodiment evaluates the robustness of a method for detecting insect-infectable cell alphaviruses.

[0076] This embodiment uses 10μL 1×107 Plasmid standards at concentrations of 1 copy / μL were added to 90μL of PBS, H5 cell lysis buffer, and SF9 cell lysis buffer, respectively, as samples for testing. Sample extraction was performed using a commercially available viral RNA extraction kit (QIAGEN GmbH, catalog number 52906). Sample detection was conducted using a commercially available qPCR premix (Roche, catalog number 04887301001) and the qPCR system and method described in Example 2. For matrix samples of the same virus, the average Cp values ​​of the PBS matrix and other matrices were calculated and differencing to assess the interference of cell matrices on sample extraction and detection results. Robustness assessment required that the absolute value of the average Cp value difference (|ΔCp|) ≤ 1.5, and the relative standard deviation (RSD) of the Cp values ​​of the corresponding matrices ≤ 5%, indicating that the cell matrix does not interfere with detection, has good stability, and is suitable for detecting such samples. The detection results are shown in Tables 12-23, demonstrating that the primer-probe combination provided by this invention is unaffected by common cell matrices during detection, exhibiting excellent robustness.

[0077] Table 12 Durability of WEEV (PBS matrix and H5 cell lysate)

[0078] Table 13 Durability of WEEV (PBS matrix and SF9 cell lysate)

[0079] Table 14 Durability of EEEV (PBS matrix and H5 cell lysate)

[0080] Table 15 Durability of EEEV (PBS matrix and SF9 cell lysate)

[0081] Table 16. Durability of SINV (PBS matrix and H5 cell lysate)

[0082] Table 17. Durability of SINV (PBS matrix and SF9 cell lysate)

[0083] Table 18. Durability of CHIKV alphavirus (PBS matrix and H5 cell lysate)

[0084] Table 19. Durability of CHIKV alphavirus (PBS matrix and SF9 cell lysate)

[0085] Table 20 Durability of AURAV (PBS matrix and H5 cell lysate)

[0086] Table 21 Durability of AURAV (PBS matrix and SF9 cell lysate)

[0087] Table 22 Durability of VEEV (PBS matrix and H5 cell lysate)

[0088] Table 23 Durability of VEEV (PBS matrix and SF9 cell lysate)

[0089] Example 5: Application of primer-probe combination detection of SINV virus infection This embodiment uses Sindbis virus (SINV) cultured in Vero cells (with a viral titer of approximately 8.38 TCID50 / mL) as the test sample. Sample extraction was performed using a commercial viral RNA extraction kit (QIAGEN GmbH, catalog number 52906). Reverse transcription was performed using a commercial reverse transcription kit (Yisheng, 11141ES60). Sample detection was conducted using a commercial qPCR premix (Roche, catalog number 04887301001) and the qPCR system and method described in Example 2 (i.e., the detection system for detecting Western equine encephalitis virus (WEEV), Eastern equine encephalitis virus (EEEV), Sindbis virus (SINV), and Chikungunya virus (CHIKV). Three independent replicate extractions were performed, with 9 replicates per well, for a total of 27 replicates. All sample results must be positive, i.e., a 100% detection rate. Simultaneously, the Cp values ​​of all replicates must meet a relative standard deviation (RSD) ≤ 5%, indicating that the virus sample can be detected repeatedly and stably. The test results are shown in Table 24. It can be seen that the primer and probe combination provided by the present invention can detect 100% of all virus samples during the test, with a Cp value RSD≤5%, and no values ​​were detected in the WEEV, EEEV and CHIKV detection channels, which shows very good stability and specificity.

[0090] Table 24 Application Examples of SINV Infection

[0091] Example 6: Primer set optimization for Eastern Equine Encephalitis Virus (EEEV) In the initial primer screening and design phase, the original upstream primer F and downstream primer R were designed for Eastern Equine Encephalitis Virus (EEEV). However, during actual validation, it was found that this primer set exhibited abnormal amplification curves in a quadruple qPCR reaction system. To address this issue, primers were redesigned for the EEEV NSP4 region using Primer Premier 5 software. Upstream primers (F1, F2, F3) and downstream primers (R1, R2) were designed and combined with the original upstream primer F and downstream primer R. The qPCR system and method described in Example 2 (i.e., the detection system for Western Equine Encephalitis Virus (WEEV), Eastern Equine Encephalitis Virus (EEEV), Sindbis Virus (SINV), and Chikungunya Virus (CHIKV)) were used to detect EEEV at a concentration of 1×10⁻⁶. 7 Plasmid standards were prepared at 1 copy / μL; two replicates were set for each primer set. The primer set with a standard "S"-shaped detection curve and a small Cp value was selected for establishing the quadruple qPCR reaction system.

[0092] Primer sequences are shown in Table 25, and detection results are as follows: Figure 13 As shown in Table 26, the detection curve of primer set F3R1 is a standard "S" shape with a small Cp value, with an average Cp value of 14.98.

[0093] Table 25 Primer sequences for Eastern Equine Encephalitis Virus (EEEV)

[0094] Table 26 Examples of primer set optimization for Eastern Equine Encephalitis Virus (EEEV)

[0095] Example 7: Optimization of primer and probe concentrations for Western Equine Encephalitis Virus (WEEV) This embodiment addresses the issue of abnormal amplification curves for Western equine encephalitis virus (WEEV) in a quadruple qPCR reaction system by optimizing primer and probe concentrations. Using the qPCR system and method from Example 2 (i.e., the detection system for Western equine encephalitis virus WEEV, Eastern equine encephalitis virus EEEV, Sindbis virus SINV, and Chikungunya virus CHIKV), the initial concentration of the Western equine encephalitis virus primer and probe working solution was uniformly set to 10 μM, along with the other viruses in the system. After application, abnormal amplification curves appeared. Based on this, the concentration of the Western equine encephalitis virus primer and probe working solution was optimized by reducing it from 10 μM to 3 μM, achieving a detection rate of 1 × 10⁻⁶ for Western equine encephalitis virus WEEV. 7 Plasmid standards were prepared at 1 copy / μL; two replicates were set for each concentration. The probe concentrations that produced the standard "S"-shaped detection curve were compared and used to establish a quadruple qPCR reaction system.

[0096] Test results as follows Figure 14 and Figure 15 As shown, reducing the primer probe concentration to 3 μM can make the detection curve exhibit a standard "S" shape.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multiplex qPCR primer-probe set for detecting alphavirus, characterized in that, It includes an upstream primer WEEV-F, a downstream primer WEEV-R, and a fluorescent probe WEEV-P for detecting western equine encephalitis virus. The nucleotide sequence of the upstream primer WEEV-F is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer WEEV-R is shown in SEQ ID NO.2, and the nucleotide sequence of the fluorescent probe WEEV-P is shown in SEQ ID NO.

3. The upstream primer EEEV-F, the downstream primer EEEV-R, and the fluorescent probe EEEV-P are used to detect eastern equine encephalitis virus. The nucleotide sequence of the upstream primer EEEV-F is shown in SEQ ID NO.4, the nucleotide sequence of the downstream primer EEEV-R is shown in SEQ ID NO.5, and the nucleotide sequence of the fluorescent probe EEEV-P is shown in SEQ ID NO.

6. The upstream primer SINV-F, the downstream primer SINV-R, and the fluorescent probe SINV-P are used for detecting Sinderby virus. The nucleotide sequence of the upstream primer SINV-F is shown in SEQ ID NO.7, the nucleotide sequence of the downstream primer SINV-R is shown in SEQ ID NO.8, and the nucleotide sequence of the fluorescent probe SINV-P is shown in SEQ ID NO.

9. The upstream primer CHIKV-F, the downstream primer CHIKV-R, and the fluorescent probe CHIKV-P for detecting Chikungunya virus are shown in SEQ ID NO.10, the nucleotide sequence of the upstream primer CHIKV-F is shown in SEQ ID NO.11, and the nucleotide sequence of the fluorescent probe CHIKV-P is shown in SEQ ID NO.

12. The upstream primer AURAV-F, the downstream primer AURAV-R, and the fluorescent probe AURAV-P for detecting AURA virus are shown in SEQ ID NO.13, the nucleotide sequence of the upstream primer AURAV-F is shown in SEQ ID NO.14, and the nucleotide sequence of the fluorescent probe AURAV-P is shown in SEQ ID NO.

15. The upstream primer VEEV-F, the downstream primer VEEV-R, and the fluorescent probe VEEV-P are used to detect Venezuelan equine encephalitis virus. The nucleotide sequence of the upstream primer VEEV-F is shown in SEQ ID NO.16, the nucleotide sequence of the downstream primer VEEV-R is shown in SEQ ID NO.17, and the nucleotide sequence of the fluorescent probe VEEV-P is shown in SEQ ID NO.

18.

2. The multiplex qPCR primer-probe set according to claim 1, characterized in that, The fluorescent probes in the primer-probe set are labeled with a fluorescent reporter group at their 5' end and a fluorescent quencher group at their 3' end.

3. The multiplex qPCR primer-probe set according to claim 2, characterized in that, The fluorescent reporter group includes any one of FAM, VIC, ROX or CY5; and / or the fluorescent quencher group includes BHQ1 or BHQ2.

4. The use of the multiplex qPCR primer-probe set according to any one of claims 1 to 3 in the preparation of a kit for detecting alphavirus.

5. A multiplex qPCR kit for detecting alphavirus, characterized in that, It includes the multiplex qPCR primer and probe set as described in any one of claims 1 to 3.

6. The multiplex qPCR kit according to claim 5, characterized in that, Based on a total volume of 25 μL, the detection system of the multiplex qPCR kit includes the following components: 1 μL of nucleic acid-free water, 13 μL of qPCR reaction solution, 0.5 μL of primer mixing working solution, 0.5 μL of probe mixing working solution, and 10 μL of template.

7. The multiplex qPCR kit according to claim 6, characterized in that, In the primer working solution, the final concentrations of upstream primer EEEV-F, downstream primer EEEV-R, upstream primer SINV-F, downstream primer SINV-R, upstream primer CHIKV-F, downstream primer CHIKV-R, upstream primer AURAV-F, downstream primer AURAV-R, upstream primer VEEV-F, and downstream primer VEEV-R are all 10 μM; the final concentrations of upstream primer WEEV-F and downstream primer WEEV-R in the primer working solution are both 3 μM. And / or, in the probe mixed working solution, the final concentrations of fluorescent probes EEEV-P, SINV-P, CHIKV-P, AURAV-P, and VEEV-P are all 10 μM; and the final concentration of fluorescent probe WEEV-P in the probe mixed working solution is 3 μM.

8. A method for detecting various alphaviruses that can infect insect cells for non-diagnostic purposes, characterized in that, The process includes the following steps: extracting RNA from the sample to be tested and reverse transcribing it into cDNA; using the cDNA as a template, performing qPCR amplification using the multiplex qPCR primer and probe set according to any one of claims 1 to 3 or the multiplex qPCR kit according to any one of claims 5 to 7; Fluorescent signals were collected, and the amplification curves and Cp values ​​were used to determine whether the samples tested were positive for Western equine encephalitis virus, Eastern equine encephalitis virus, Sindbis virus, Chikungunya virus, AURA virus, and Venezuelan equine encephalitis virus.

9. The method according to claim 8, characterized in that, The criteria for judgment are as follows: if the Cp value of the sample to be tested is less than 40 and there is an obvious amplification curve, it is judged as positive; if the Cp value of the sample to be tested is greater than or equal to 40 or there is no obvious amplification curve, it is judged as negative.

10. The method according to claim 8, characterized in that, The qPCR reaction program is as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 60℃ annealing for 1 min, 45 cycles; 40℃ cooling for 30 s.

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