Double-RPA-LFA human herpes virus visual detection method, primer probe and application

Abstract

The invention discloses a double-RPA-LFA human herpes virus visual detection method, a primer probe and application, and relates to the technical field of multiple herpes virus detection. The invention provides a primer probe combination for dual detection of HCMV and HHV-6 based on RPA-LFA, and also provides a corresponding detection method and a detection product. According to the detection method for double detection of the human herpes viruses HCMV and HHV-6 and the primer and probe combination, the false positive problem can be effectively avoided, dimer or cross reaction cannot be generated between the primer and the probe of the two viruses, and interference cannot be generated; the detection sensitivity is very high, and the lower detection limit reaches 1 * 10 < 3 > copies / 50 microliters.

Description

Technical Field

[0001] This invention relates to the field of multiplex herpesvirus detection technology, and particularly to a visual detection method, primers, and applications for human herpesvirus using dual RPA-LFA. Background Technology

[0002] Recombinase polymerase amplification (RPA), as an isothermal amplification technique, can achieve rapid nucleic acid amplification at 37-42℃. Lateral flow chromatography (LFA), with its ease of operation and visualized results, has been widely used in point-of-care testing. Currently, numerous studies combine LPA and LFA techniques for the detection of target genes. However, existing RPA-LFA techniques have some significant drawbacks. Most studies can only detect single viruses, failing to meet the need for simultaneous detection of multiple viruses. Detecting multiple viruses requires different primers; however, these different primers are prone to dimerization or cross-reaction in the reaction system, leading to non-specific color development in the amplification tube, resulting in false positives and affecting detection accuracy. In multiplex detection, signal interference easily occurs between multicolor labeled probes, making accurate interpretation of results difficult.

[0003] There are eight types of human herpesviruses (HHVs). Among them, human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6) belong to the β-herpesvirus subfamily, and their genomes share a similarity of 40%-50%. This high homology presents technical challenges for simultaneous detection. Co-infection of HCMV and HHV-6 is not uncommon in clinical settings, especially in immunocompromised individuals (such as organ transplant recipients and HIV patients). Co-infection can exacerbate the condition, leading to more serious complications (such as pneumonia, encephalitis, and bone marrow suppression), significantly increasing the difficulty of clinical diagnosis and treatment. Currently, there are no commercially available RPA-LFA products for simultaneous detection of these two viruses. Primary healthcare units mainly rely on laboratory nucleic acid testing when detecting these two viruses. This method requires specialized technicians and sophisticated instruments, is complex to operate, and has a long testing time, failing to meet the needs of rapid point-of-care diagnosis and hindering the timely identification of co-infection cases to guide clinical decision-making.

[0004] Therefore, developing a visual rapid detection system that can accurately distinguish between HCMV and HHV-6 is of great clinical significance for improving the detection efficiency of single and synergistic infections in primary healthcare units and for achieving early diagnosis and timely prevention and control of related diseases. Summary of the Invention

[0005] This invention provides a visual detection method, primers, probes, and applications for human herpesvirus using dual RPA-LFA, specifically implemented through the following techniques.

[0006] This invention provides an application of a dual RPA-LFA human herpesvirus detection system. The system is used to detect human cytomegalovirus (CMV) and human herpesvirus 6 (HHV-6) without the aim of disease diagnosis or treatment. The system includes an HCMV detection primer pair, an HHV-6 detection primer pair, an HCMV detection probe, and an HHV-6 detection probe. The nucleotide sequences of the HCMV detection primer pair are shown in SEQ ID NO. 1 and 2, the nucleotide sequences of the HHV-6 detection primer pair are shown in SEQ ID NO. 11 and 12, the nucleotide sequence of the HCMV detection probe is shown in SEQ ID NO. 36, and the nucleotide sequence of the HHV-6 detection probe is shown in SEQ ID NO. 34. The HHV-6 detection probe has a first fluorescent group at its 5' end and a second fluorescent group at its 5' end, with the first and second fluorescent groups emitting different fluorescence. The HHV-6 detection probe and its 3' segment have blocking groups and internal debasement sites.

[0007] Furthermore, the concentrations of the HCMV detection primer pair and the HHV-6 detection primer pair are 400-1200 nM.

[0008] Furthermore, the concentration of the HCMV detection primer pair is 800 nM, and the concentration of the HHV-6 detection primer pair is 400 nM.

[0009] Furthermore, the total amount of the HCMV detection primer pair and the HHV-6 detection primer pair used is 4-6 μL.

[0010] Furthermore, the total amount of the HCMV detection primer pair and the HHV-6 detection primer pair used is 5 μL.

[0011] Furthermore, the amplification reaction time of the RPA is 5-20 min, and the amplification reaction temperature is 35-43℃.

[0012] This invention provides a primer-probe combination for human herpesvirus detection based on dual RPA-LFA, comprising an HCMV detection primer pair, an HHV-6 detection primer pair, an HCMV detection probe, and an HHV-6 detection probe. The nucleotide sequences of the HCMV detection primer pair are shown in SEQ ID NO. 1 and 2, the nucleotide sequences of the HHV-6 detection primer pair are shown in SEQ ID NO. 11 and 12, the nucleotide sequence of the HCMV detection probe is shown in SEQ ID NO. 36, and the nucleotide sequence of the HHV-6 detection probe is shown in SEQ ID NO. 34. The 5' end of the HHV-6 detection probe has a first fluorescent group and the 5' end of the HHV-6 detection probe has a second fluorescent group, the fluorescence emitted by the first fluorescent group and the second fluorescent group are different. The 3' segment of the HHV-6 detection probe and the HHV-6 detection probe have blocking groups and debasement sites inside.

[0013] The present invention also provides a human herpesvirus detection product based on dual RPA-LFA, comprising the above-mentioned primer-probe combination.

[0014] Furthermore, the human herpesvirus detection product is a detection kit, which includes RPA amplification reagent and lateral flow chromatography test strips.

[0015] Furthermore, the RPA amplification reagent includes nfo enzyme, nfo enzyme buffer, ddH2O, TwistAmp® Basickit, and test strip diluent.

[0016] Furthermore, the RPA buffer contains 0.5% Tween-20 by mass.

[0017] This invention also provides a dual RPA-LFA method for detecting human herpesviruses that is not intended for disease diagnosis or treatment. The method uses the aforementioned primer-probe combination or any of the aforementioned human herpesvirus detection products for detection; and includes the following steps:

[0018] DNA was extracted from the sample to be tested and RPA amplification was performed; the RPA amplification products were then detected by lateral flow chromatography.

[0019] Compared with existing technologies, the advantages of this invention are as follows: Based on recombinase polymerase amplification technology and lateral flow chromatography technology, this invention provides a detection method and primer-probe combination for dual detection of human herpesviruses HCMV and HHV-6, which can effectively avoid false positives, and there is no interference between the primers and probes for HCMV and HHV-6; it has very high detection sensitivity, with a detection limit of 1×10⁻⁶. 3 Copy / 50μL. Attached Figure Description

[0020] Figure 1 These are preliminary results from an initial trial of the RPA-LFA detection system. The first image from the left shows HHV-8 positivity at T4; the second image from the left shows HHV-6 positivity at T3; the second image from the right shows HCMV positivity at T2; and the first image from the right shows HHV-7 positivity at T1.

[0021] Figure 2 The results of HHV-8 probe screening are shown. BCBL-1 is an HHV-8 positive cell; HUV-EC-C is an HHV-6 positive cell; B95-8 is an EBV positive cell; and HeLa cells are the negative group. It can be seen that HHV-8 positivity shows staining at T4; the negative group and the blank control group only show staining at the C line.

[0022] Figure 3 The results of HHV-6 probe screening are shown. BCBL-1 is an HHV-8 positive cell; HUV-EC-C is an HHV-6 positive cell; B95-8 is an EBV positive cell; and HeLa cells are the negative group. It can be seen that HHV-6 positivity shows staining at T3; the negative group and the blank control group only show staining at the C line.

[0023] Figure 4 The results show the screening results for HCMV probes. HCMV positive cells showed staining at T2 chromogenicity; the negative group and blank control group showed staining only at the C line; BCBL-1 were HHV-8 positive cells; HUV-EC-C were HHV-6 positive cells; B95-8 were EBV positive cells; and HeLa cells were in the negative group.

[0024] Figure 5 The results show the screening results for the HHV-7 probe. HHV-7 positive cells showed staining at T1 chromogenicity; the negative group and the blank control group showed staining only at the C-line; HeLa cells were in the negative group.

[0025] Figure 6 The results of the attempt to construct a multiplex RPA-LFA detection system are shown. In this system, test strip 1 represents a mixture of three viral plasmids: HHV-8, HHV-6, and HCMV; test strip 2 represents a mixture of HHV-8 and HHV-6 plasmids; test strip 3 represents a mixture of HHV-8 and HCMV plasmids; and test strip 4 represents a mixture of HHV-6 and HCMV plasmids.

[0026] Figure 7The results show the optimized primer concentrations for the dual RPA-LFA detection system. In the left figure, strip 1 indicates that the primer concentrations for both HHV-6 and HCMV are 400 nM; strip 2 indicates that the primer concentrations for HHV-6 and HCMV are 800 nM and 400 nM, respectively; and strip 3 indicates that the primer concentrations for HHV-6 and HCMV are 1200 nM and 400 nM, respectively. In the right figure, strip 1 indicates a total primer volume of 4 μL; strip 2 indicates a total primer volume of 5 μL; and strip 3 indicates a total primer volume of 6 μL.

[0027] Figure 8 The results show the optimized probe concentrations for the dual RPA-LFA detection system. In the left figure, strip 1 indicates that both HHV-6 and HCMV probe concentrations are 60 nM; strip 2 indicates that HHV-6 and HCMV probe concentrations are 120 nM and 60 nM, respectively; and strip 3 indicates that HHV-6 and HCMV probe concentrations are 180 nM and 60 nM, respectively. In the right figure, strip 1 indicates a total probe volume of 0.4 μL; strip 2 indicates a total probe volume of 0.6 μL; strip 3 indicates a total probe volume of 0.8 μL; and strip 4 indicates a total probe volume of 1 μL.

[0028] Figure 9 The results show the optimization of temperature and time for the dual RPA-LFA detection system. The left figure shows the optimized reaction time for the dual RPA-LFA detection system; the right figure shows the optimized reaction temperature for the dual RPA-LFA detection system.

[0029] Figure 10 This presents the sensitivity study results of the dual RPA-LFA detection system. The mixed plasmid represents the combination of HHV-6 and HCMV plasmids; the mixed primers represent the combination of HHV-6 and HCMV primers. It can be seen that HHV-6 positivity develops at T3; HCMV positivity develops at T2.

[0030] Figure 11 This demonstrates the application of the dual RPA-LFA system in cell detection. HUV-EC-C cells are HHV-6 positive; HeLa cells are the negative group. HHV-6 positivity is observed to show staining at T3; HCMV positivity shows staining at T2. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] 1. Experimental Materials

[0033] In the specific embodiments provided by this invention, the *E. coli* strain DH5α and the pMD18-T vector were deposited and provided by our laboratory. All cell lines and live HHV-8 virus were deposited and provided by the China Center for Type Culture Collection (CCTCC), Wuhan University. The cell lines used in the experiments are shown in Table 1 below.

[0034] Table 1. Cell lines used in the experiment

[0035]

[0036]

[0037] 2. Main reagents for the experiment

[0038] The main reagents required for the experiment are shown in Table 2 below.

[0039] Table 2 Main reagents required for the experiment

[0040]

[0041] The dual RPA-LFA human herpesvirus detection system provided by this invention uses a lateral flow chromatography test strip whose structure includes a sample pad, a conjugate pad, a chromatographic membrane, and an absorbent pad (Wick), as well as a backing card.

[0042] The sample pad is the starting end of the test strip, used to absorb the sample and uniformly transfer the liquid to the binding pad through capillary action. The sample pad is typically made of hydrophilic treated glass fiber or cellulose membrane to ensure rapid absorption and stable liquid flow. The absorbent pad is located at the end of the test strip, maintaining the balance of capillary forces during chromatography, promoting sample flow along the strip, preventing backflow, and absorbing excess liquid. The absorbent pad is usually made of highly absorbent cardboard or cellulose material and needs to be tightly bonded to the chromatographic membrane to ensure continuous flow. The support plate's function is to fix the layers in place, especially ensuring precise alignment and adhesion between layers to prevent interlayer leakage or flow obstruction, thus maintaining the structural integrity of the test strip.

[0043] The conjugate pad of this invention adsorbs labeled bioactive materials; for example, it adsorbs colloidal gold-labeled antibodies. As the sample flows through, these labels specifically bind to the target analytes in the sample, forming complexes that can be captured by subsequent steps. In the dual-lateral flow chromatography test strip for HCMV and HHV-6 of this invention, the conjugate pad contains multiple labels targeting these two viruses, enabling simultaneous detection of multiple targets.

[0044] The chromatographic membrane is the core component of the entire test strip, containing the test line (T line) and control line (C line), providing a crucial platform for the reaction of the analytes. The T line is coated with capture antibodies or antigens, which specifically capture the complex of the target analyte and the label, resulting in color development. The C line embeds anti-labeled antibodies (such as secondary antibodies) to verify the effectiveness of the test strip; the C line should develop color regardless of the presence of the target analyte. In this invention, when performing dual detection, there will be two T lines on the chromatographic membrane, targeting HCMV and HHV-6 analytes respectively.

[0045] Example 1: Cell culture, resuscitation, passage, cryopreservation, and DNA extraction

[0046] 1. Cell culture: Cell culture in this example is performed according to the routine experimental methods summarized by the China Center for Type Culture Collection (CCTCC).

[0047] 2. Cell resuscitation: Thaw the cell samples, centrifuge, resuspend and culture them, and place the culture flasks in a 37°C, 5% CO2 incubator.

[0048] 3. DNA extraction

[0049] (1) Extraction of cell line DNA

[0050] Select cells in good growth condition, digest them with trypsin, centrifuge, and gently disperse them to form a cell suspension. Perform the extraction procedure according to the instructions of the blood / cell / tissue genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. Set aside the collected cellular DNA and label it in EP tubes.

[0051] To facilitate subsequent experiments, the concentration of extracted DNA can be determined using a NanoDrop micro spectrophotometer, diluted to 50 ng / μL with ddH2O, and stored at -20°C for later use.

[0052] (2) Viral DNA extraction

[0053] The HCMV viral solution preserved at the China Center for Type Culture Collection (CCTCC) was processed according to the instructions of the OMEGA Viral DNA Kit to remove other impurities and obtain viral DNA. The viral DNA was then extracted and stored at -20°C for later use.

[0054] Example 2: Construction of a Dual RPA-LFA Detection System

[0055] 1. Design of RPA detection primers

[0056] In the initial stages of the study, to achieve simultaneous detection of HCMV, HHV-6, HHV-7, and HHV-8, multiple candidate primer pairs were designed around the conserved gene regions of the four viruses. Multiple reference sequences of the four viruses were retrieved from the NCBI database, and sequence alignment was performed using ClustalX to screen regions with no continuous base variations of more than 200 bp as targets, ensuring the primers' universality across different strains of the same virus. Simultaneously, BLAST whole-genome alignment was used to strictly control the sequence similarity of candidate target regions among the four viruses to <60%, and the primer binding region to have ≥5 consecutive base differences, avoiding cross-amplification at the sequence level. For example, the HCMV primer targets the UL83 gene, with only 41% similarity to the corresponding region of HHV-7; the HHV-8 primer targets the ORF75 gene, with less than 39% homology to HHV-6, laying the foundation for subsequent specific detection.

[0057] Finally, HCMV gene (NC_006273.2), HHV-6 gene (NC_001664.4), HHV-7 gene (NC_001716.2), and HHV-8 gene (NC_009333.1) were selected, and a total of 50 primer pairs were designed. Then, multiple rounds of design optimization were carried out, and 15 pairs of highly specific RPA detection primers were screened for further screening, as shown in Table 3-6 below.

[0058] Table 3. RPA detection primer sequences for screening HCMV (NC_006273.2)

[0059]

[0060] Table 4. Primer sequences for RPA detection of HHV-6 (NC_001664.4)

[0061]

[0062]

[0063] Table 5. Primer sequences for RPA detection of HHV-7 (NC_001716.2)

[0064]

[0065] Table 6. Primer sequences for RPA detection of HHV-8 (NC_009333.1)

[0066]

[0067]

[0068] As shown in Table 3-6, there are 2 pairs of primers for the HCMV gene (NC_006273.2), 5 pairs of primers for the HHV-6 gene (NC_001664.4), 3 pairs of primers for the HHV-7 gene (NC_001716.2), and 5 pairs of primers for the HHV-8 gene (NC_009333.1).

[0069] 2. Screening of detection primers

[0070] From Table 5-8 above, primers for RPA detection targeting four human herpesviruses, namely HCMV, HHV-6, HHV-7, and HHV-8, were finally selected.

[0071] Specifically, the primer pair nucleotide sequences for HCMV (NC_006273.2) are shown in SEQ ID NO. 1 and 2, the primer pair nucleotide sequences for HHV-6 (NC_001664.4) are shown in SEQ ID NO. 11 and 12, the primer pair nucleotide sequences for HHV-7 (NC_001716.2) are shown in SEQ ID NO. 15 and 16, and the primer pair nucleotide sequences for HHV-8 (NC_009333.1) are shown in SEQ ID NO. 29 and 30.

[0072] 3. An attempt to construct a multi-RPA-LFA detection system

[0073] While conventional PCR and RPA detection systems can effectively detect human herpesviruses, both require agarose gel electrophoresis after amplification to visualize the results. This electrophoresis process is not only time-consuming but also carries the risk of contamination. Lateral Flow Assay (LFA), a lateral flow chromatography technique, is not only simple to operate but also rapid and efficient. Therefore, this embodiment considers combining multiplex RPA with LFA to establish a rapid and visualized detection system for the simultaneous detection of multiple human herpesviruses.

[0074] We fluorescently labeled the selected primers for detecting HCMV, HHV-6, HHV-7, and HHV-8. Specifically, the forward primer for HHV-8 was labeled with FITC, the forward primer for HHV-6 with TAMRA, the forward primer for HCMV with Digoxin, and the forward primer for HHV-7 with Cy5. The reverse primers for all four viruses were labeled with Biotin.

[0075] Because the RPA-LFA system is highly prone to false positives, each of the labeled primers needs to be verified individually. Specifically, in this embodiment, the corresponding viral template is added to the labeled forward and reverse primers, and the amplification reaction is carried out at 41°C for 20 min to amplify products containing both labels. The RPA reaction system uses the TwistAmp® Basickit, as shown in Table 7 below. The sequences shown in SEQ ID NO.39-42 are selected as the target fragments in the DNA templates of HHV-8, HHV-6, HCMV, and HHV-7, respectively. Then, 1 μL of the product is added to 49 μL of lateral flow chromatography test strip dilution buffer (PBS) and mixed thoroughly (dilution ratio 1:50), and then added dropwise to the test strip; the mixture is allowed to stand at room temperature for 3 min, and the results are observed.

[0076] Table 7 RPA Reaction System

[0077]

[0078] The results are as follows Figure 1 As shown, the test strips for all four human herpesvirus positive samples showed positive results, but the corresponding test lines for the negative control group and the ddH2O blank control group also showed positive bands. After analysis, we hypothesize that primer dimers produced false positive bands on the test strips.

[0079] 4. Design and screening of different viral probes

[0080] To avoid the aforementioned false positives, we introduced detection probes into the reaction system. Since false positives may still occur after introducing probes, we designed two detection probes with corresponding labels for each of the four viruses—HHV-8, HHV-6, HCMV, and HHV-7—and performed corresponding screening.

[0081] The probe sequence precisely matches the non-homologous conserved regions of the virus and has been verified by BLAST to have no cross-binding with other herpesviruses. Even with a viral-to-template concentration ratio of 10:1, specific color development can still be achieved through probe labeling differences, solving the signal interference problem during co-detection of homologous viruses. Simultaneously, the binding regions of the primers and probes are spaced 15-20 bp apart to avoid competitive binding during RPA amplification. The biotin labeling site of the downstream primer is 10 bp from the 3' end, and the THF site of the probe is ≥30 bp from the 5' end, ensuring efficient nfoase cleavage and the integrity of dual labeling of the amplification product, resulting in a 40% improvement in amplification efficiency compared to conventional designs.

[0082] The nucleotide sequences of the detection probe and primers are shown in Table 8 below, and both were synthesized by Tianyi Huiyuan Company. The synthesized probe and primers were prepared into a 100 μM primer solution using ddH2O and stored at -20℃.

[0083] Table 8. Nucleotide sequences of the preliminarily designed RPA detection primers and probes.

[0084]

[0085]

[0086] As shown in Table 8 above, during RPA amplification, diluted unlabeled forward primers, biotin-labeled reverse primers, and nfo enzyme and nfo enzyme buffer were added to the system. Each detection probe contains an internal abasic site in tetrahydrofuran (THF). After nfo enzyme digestion, an extendable 3'-OH group is exposed, allowing for further amplification and the generation of the target band containing both labels. Therefore, probes, nfo enzyme, and nfo enzyme buffer need to be added to the RPA system; and unlabeled forward primers and biotin-labeled reverse primers are used as controls. The reaction was carried out at 41℃ for 20 min. In addition, we added 0.5% Tween-20 to the standard RPA buffer to reduce the non-specific adsorption of primers and probes, solving the sensitivity decrease problem in direct detection of complex samples. This is the first time this has been added in RPA applications. The RPA reaction system is shown in Table 9 below.

[0087] Table 9 RPA Reaction System

[0088]

[0089]

[0090] After amplification, the amplification product and diluent were mixed at a dilution ratio of 1:50. 50 μL of the diluted reaction solution was then dropped onto the sample area of ​​the side-flow chromatography test strip. The results were observed after standing for 3 minutes.

[0091] For HHV-8, we designed two probes with FITC markings. The color development results of the test strips are as follows: Figure 2 As shown, it was clearly observed that false positives persisted even after the introduction of probe 8-Probe-1. Therefore, we redesigned probe 8-Probe-2, and found that the test strips containing HHV-8 plasmid and BCBL-1 (HHV-8) cell DNA amplification products were positive, while the HeLa negative group, HUV-EC-C (HHV-6) group, B95-8 (EBV) group, and ddH2O blank group all showed negative results. Therefore, the introduction of probe 8-Probe-1 resolved the false positive problem and made the test results more reliable.

[0092] For HHV-6, we designed two probes labeled with TAMRA. The reaction is as follows: Figure 3 As shown, adding probe 6-Probe-1 to the system did not solve the false positive problem, while the introduction of probe 6-Probe-2 avoided false positives. Specifically, the addition of HHV-6 plasmid group and HUV-EC-C (HHV-6) cell DNA group resulted in positive results after color development on the test strip, while BCBL-1 group (HHV-8), B95-8 group (EBV), HeLa negative group and ddH2O blank group all resulted in negative results. Therefore, probe 6-Probe-2 is usable.

[0093] For HCMV, we designed two probes labeled with Digoxin based on the same principle. Figure 4 The results showed that, unlike the frequent false positives, false negatives occurred after the introduction of probe 5-Probe-1. That is, all samples, including those containing positive samples, HeLa negative samples, and ddH2O blank samples, were negative. It is speculated that the addition of probe 5-Probe-1 interfered with the normal reaction process. Therefore, we redesigned probe 5-Probe-2. The results showed that the HCMV viral DNA group was positive, and all other groups were negative, as expected. Therefore, probe 5-Probe-2 is usable.

[0094] For HHV-7, we designed two probes labeled with Cy5. However, as... Figure 5 As shown, neither probe solved the false positive problem. Therefore, it is necessary to continue to change probes in the future. However, the problem is that the target fragment amplified by the HHV-7 primers is only 114 bp, the range of probe design is small, and changing probes is difficult. Therefore, further exploration is needed.

[0095] 5. Attempts to construct triple and dual RPA-LFA systems

[0096] Based on preliminary experiments, we screened out effective probes for HHV-8, HHV-6, and HCMV viruses, each with different labels. Therefore, we attempted to mix these three probes and add them to the RPA system to establish a triple RPA-LFA detection system. Simultaneously, we also combined HHV-8, HHV-6, and HCMV probes in pairs to establish a dual RPA-LFA detection system.

[0097] The results are as follows Figure 6As shown, when the three probes were mixed simultaneously, no bands were displayed on the test strips of the first experimental group. It is speculated that the system composition was too complex, causing interference between them and preventing the reaction from proceeding normally. The HHV-8 and HHV-6 mixed group had almost no bands, and the HHV-8 and HCMV mixed group also had no bands. Only the HHV-6 and HCMV mixed group, i.e., the third experimental group, showed color development, but the corresponding bands were all weak. It is speculated that the concentration and ratio of the probes and primers were not optimal.

[0098] Therefore, the present invention further optimizes the established dual RPA-LFA detection system for detecting HHV-6 and HCMV viruses, as detailed in Example 3.

[0099] Example 3: Optimization of the Dual RPA-LFA Human Herpesvirus Detection System

[0100] To further improve the detection performance of this dual RPA-LFA, we optimized the system in four aspects: primer ratio and dosage (concentration), probe ratio and dosage (concentration), reaction time, and reaction temperature. Conventional multiplex RPA-LFA systems often use an equal primer / probe design, which can easily lead to differences in sensitivity due to amplification competition. The system of this invention forms a unique reaction equilibrium mechanism through a nonlinear optimization scheme screened by orthogonal experiments.

[0101] 1. Optimization of the ratio and amount of detection primers

[0102] In this embodiment, three sets of experiments were conducted to optimize primer ratios: the concentrations of HHV-6 and HCMV primers were both 400 nM (amount / concentration ratio of 1:1), 800 nM and 400 nM (amount / concentration ratio of 2:1), and 1200 nM and 400 nM (amount / concentration ratio of 3:1). The total volume of the mixed probe was then adjusted to 4 μL, 5 μL, and 6 μL, respectively.

[0103] This embodiment optimized the ratio of the two primer pairs, set three different primer concentrations, and fixed other reaction conditions for amplification. The color development results of the test strip are as follows: Figure 7 As shown in the left figure, it can be seen that the second experimental group had the best results. Therefore, this primer ratio was selected in subsequent experiments, and the primer sets were prepared by mixing equal volumes.

[0104] This embodiment designed three sets of experiments to explore the optimal primer dosage, and the results are as follows: Figure 7 As shown in the right figure, the second experimental group showed the best amplification effect. Increasing the primer amount further affected the amplification. Therefore, the optimal total primer amount for the system was finally determined to be 5 μL, at which point the final primer concentrations of HHV-6 and HCMV in the system were 1000 nM and 500 nM, respectively.

[0105] The primers for HHV-6 and HCMV are set at the concentration ratios mentioned above, which can offset the high abundance advantage of HCMV templates commonly found in cell samples. For example, HCMV can still be detected simultaneously when the copy number is 5 times that of HHV-6, thus solving the problem of missed detection caused by differences in the initial template amount of homologous viruses.

[0106] 2. Optimization of detection probes and dosage

[0107] In this embodiment, the concentrations of the detection probes for HHV-6 and HCMV were set at 60 nM (amount / concentration ratio of 1:1), 120 nM and 60 nM (amount / concentration ratio of 2:1), and 180 nM and 60 nM (amount / concentration ratio of 2:1). The total amount of mixed primers was then adjusted.

[0108] Since the probes are crucial in this system, we optimized their ratio and dosage. In this embodiment, the ratio of the two probe groups was adjusted, and three experimental groups were set up for amplification. The color development results of the test strips are as follows... Figure 8 As shown in the left figure, the optimal probe ratio for the second experimental group is 2:1.

[0109] After determining the probe ratio, we adjusted the total amount of probes used and designed four sets of experiments. The color development results of the test strips are as follows: Figure 8 As shown in the right figure, it is clear that the colorimetric results of the second experimental group were the easiest to observe. As the total amount of probe continued to increase, the colorimetric results gradually weakened. Therefore, the probe amount of the second experimental group was selected in subsequent experiments, at which point the final concentrations of HHV-6 and HCMV probes in the system were 120 nM and 60 nM, respectively.

[0110] 3. Optimization of amplification reaction time and reaction temperature

[0111] This embodiment also investigated the optimal reaction temperature and optimal reaction time of the system; five temperature gradients were set: 35℃, 37℃, 39℃, 41℃, and 43℃, and amplification was performed for 5 min, 10 min, 15 min, and 20 min after fixing the temperature. The amplified product was mixed with the diluent at a ratio of 1:50, and then 50 μL was added dropwise to the sample area of ​​the side-flow chromatography strip. After standing for 3 min, the results were observed.

[0112] The color development results of the test strip with optimized amplification time are as follows: Figure 9 As shown in the left image, the color development results are sufficiently clear after 15 minutes of reaction.

[0113] The color development results of the test strip optimized for amplification temperature are as follows: Figure 9As shown in the right figure, the test strip shows the best color development effect when the reaction temperature of the system is 39℃, so 39℃ is determined as the optimal reaction temperature.

[0114] Example 4: Sensitivity determination of the dual RPA-LFA human herpesvirus detection system

[0115] In this embodiment, to investigate the detection sensitivity of the dual RPA-LFA detection system, the concentrations of HHV-6 and HCMV plasmids were diluted to 1×10⁻⁶. 8 1×10 7 1×10 6 1×10 5 1×10 4 1×10 3 1×10 2 and 1×10 1 Copy number / μL.

[0116] This embodiment explores the detection limits of the dual RPA-LFA detection system for simultaneously detecting HHV-6 and HCMV, as well as for detecting HHV-6 and HCMV individually.

[0117] The results are as follows Figure 10 As shown, it can be clearly observed that the limit of detection (LOD) of this dual RPA-LFA detection system (50 μL) is 1 × 10⁻⁶ for both detecting a single virus and simultaneously detecting HHV-6 and HCMV viruses. 3 Copy number / 50 μL (20 copies / μL). This indicates that the dual RPA-LFA detection system achieves rapid result visualization while also ensuring high-sensitivity detection performance.

[0118] Application Example: Application of the Dual RPA-LFA Human Herpesvirus Detection System in Cell Detection

[0119] In this application example, 10 negative cells previously tested were randomly selected, and HCMV viral DNA and HUV-EC-C (HHV-6) cell DNA were re-extracted and detected using a dual RPA-LFA system. The results after color development on the test strip are as follows: Figure 10 As shown, the detection results of negative cells were all negative, consistent with the results of the established eight-fold PCR system and four-fold RPA system; while HCMV viral DNA showed color development at T2, and HUV-EC-C cells, which are HHV-6 positive cells, showed color development at T3, both consistent with expectations. This indicates that the dual RPA-LFA detection system not only achieves rapid visualization of results but also ensures the accuracy of the detection results.

[0120] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An application of a dual RPA-LFA human herpesvirus detection system, characterized in that, The human herpesvirus detection system is used to detect human cytomegalovirus (CMV) and human herpesvirus 6 (HHV-6) without the purpose of disease diagnosis or treatment. The system includes HCMV detection primer pairs, HHV-6 detection primer pairs, an HCMV detection probe, and an HHV-6 detection probe. The nucleotide sequences of the HCMV detection primer pairs are shown in SEQ ID NO. 1 and 2, the nucleotide sequences of the HHV-6 detection primer pairs are shown in SEQ ID NO. 11 and 12, the nucleotide sequences of the HCMV detection probe are shown in SEQ ID NO. 36, and the nucleotide sequences of the HHV-6 detection probe are shown in SEQ ID NO.

34. The HHV-6 detection probe has a first fluorescent group at its 5' end and a second fluorescent group at its 5' end, with the first and second fluorescent groups emitting different fluorescence. The HHV-6 detection probe and its 3' segment have blocking groups and internal debasement sites.

2. The application of the dual RPA-LFA human herpesvirus detection system according to claim 1, characterized in that, The concentrations of the HCMV detection primer pairs and HHV-6 detection primer pairs are 400-1200 nM; Furthermore, the concentration of the HCMV detection primer pair is 800 nM, and the concentration of the HHV-6 detection primer pair is 400 nM.

3. The application of the dual RPA-LFA human herpesvirus detection system according to claim 1, characterized in that, The total amount of the HCMV detection primer pair and the HHV-6 detection primer pair used is 4-6 μL; Furthermore, the total amount of the HCMV detection primer pair and the HHV-6 detection primer pair used is 5 μL.

4. The application of the dual RPA-LFA human herpesvirus detection system according to claim 1, characterized in that, The amplification reaction time of the RPA is 5-20 min, and the amplification reaction temperature is 35-43℃.

5. A primer-probe combination for detecting human herpesviruses based on dual RPA-LFA, characterized in that, The device includes an HCMV detection primer pair, an HHV-6 detection primer pair, an HCMV detection probe, and an HHV-6 detection probe. The nucleotide sequences of the HCMV detection primer pair are shown in SEQ ID NO. 1 and 2, the nucleotide sequences of the HHV-6 detection primer pair are shown in SEQ ID NO. 11 and 12, the nucleotide sequence of the HCMV detection probe is shown in SEQ ID NO. 36, and the nucleotide sequence of the HHV-6 detection probe is shown in SEQ ID NO.

34. The 5' end of the HHV-6 detection probe has a first fluorescent group and the 5' end of the HHV-6 detection probe has a second fluorescent group. The fluorescence emitted by the first fluorescent group and the second fluorescent group are different. The 3' segment of the HHV-6 detection probe and the HHV-6 detection probe has a blocking group and a debasement site inside.

6. A human herpesvirus detection product based on dual RPA-LFA, characterized in that, Includes the primer-probe combination as described in claim 5.

7. The human herpesvirus detection product based on dual RPA-LFA according to claim 6, characterized in that, The test kit includes RPA amplification reagents and lateral flow chromatography test strips.

8. The human herpesvirus detection product based on dual RPA-LFA according to claim 7, characterized in that, The RPA amplification reagents include nfo enzyme, nfo enzyme buffer, ddH2O, TwistAmp® Basic kit, and test strip diluent.

9. The human herpesvirus detection product based on dual RPA-LFA according to claim 7, characterized in that, The RPA buffer contains 0.5% Tween-20 by mass.

10. A dual RPA-LFA method for detecting human herpesviruses not intended for disease diagnosis or treatment, characterized in that, The detection is performed using the primer-probe combination as described in claim 5, or using the human herpesvirus detection product as described in any one of claims 6-8; including the following steps: DNA was extracted from the sample to be tested and RPA amplification was performed; the RPA amplification products were then detected by lateral flow chromatography.

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