Quadruple RPA (recombinase polymerase amplification) detection system for human herpes virus based on isothermal amplification and application of quadruple RPA detection system

Abstract

The invention discloses a human herpes virus quadruple RPA (recombinase polymerase amplification) detection system based on isothermal amplification and application of the human herpes virus quadruple RPA detection system, and relates to the technical field of multiple human herpes virus RPA detection. The quadruple RPA detection system for the human herpes virus provided by the invention comprises a group A detection primer combination or a group B detection primer combination, the group A detection primer combination comprises detection primer pairs for respectively detecting HSV-1, HSV-2, VZV and EBV, and the group B detection primer combination comprises detection primer pairs for respectively detecting HCMV, HHV-6, HHV-7 and HHV-8. The invention further provides a quadruple RPA detection method for the human herpes virus based on the system. The quadruple RPA detection system provided by the invention has the advantages that the detection time is greatly shortened, the detection sensitivity is high, the specificity is strong, the carrying is more convenient, the required equipment is simple, and greater convenience is provided for human herpes virus detection.

Description

Technical Field

[0001] This invention relates to the field of multiple human herpesvirus RPA detection technology, and particularly to a human herpesvirus quadruple RPA detection system based on isothermal amplification and its application. Background Technology

[0002] Currently, eight human herpesviruses (HHVs) have been identified: herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus type 6 (HHV-6), human herpesvirus type 7 (HHV-7), and human herpesvirus type 8 (HHV-8). Existing methods for detecting human herpesviruses (such as virus isolation and culture, serum detection, etc.) suffer from insufficient sensitivity, complex operation, and limited multiplexing capabilities, making it difficult to meet the demand for simultaneous and rapid detection of multiple viruses in complex samples.

[0003] Recombinase polymerase amplification (RPA), as an isothermal amplification technique, can achieve rapid nucleic acid amplification at 37-42℃. However, the realization of multiplex RPA detection faces significant technical obstacles, primarily due to the complexity of its reaction system. The RPA system is not simply a mixture of enzymes and primers; it contains multiple key proteins, including recombinases, single-stranded binding proteins, and DNA polymerases. The concentrations and ratios of each component must be strictly matched—the recombinase needs to bind to the primers to form a complex to initiate amplification, the single-stranded binding protein needs to stabilize the single-stranded template, and the polymerase is responsible for strand extension. These components are interdependent and mutually restrictive. When multiple primer pairs are introduced for multiplex detection, not only is primer dimer formation likely, but competition for binding between different components and primers intensifies. Some primers may preferentially bind to the recombinase, monopolizing reaction resources, while the amplification of other primers is significantly inhibited, ultimately resulting in severely uneven amplification efficiency. This complex interaction within the system limits the current upper limit of multiplex RPA detection to three-fold, and there is still no mature system for multiple types of the same major pathogen class (such as human herpesvirus). Therefore, it is urgent to develop a multi-level RPA system that can quickly and accurately detect multiple HHVs. Summary of the Invention

[0004] Existing RPA systems contain multiple interacting components such as recombinases, single-stranded binding proteins, and DNA polymerases. These components are functionally dependent and highly competitive, making multiplex detection extremely difficult. Not only is multiplex detection for the same class of pathogens (such as human herpesviruses) completely lacking, but even for different types of pathogens, the upper limit of existing multiplex RPA systems is only three-fold, with no successful cases of quadruple detection. This invention provides a quadruple RPA detection system for human herpesvirus based on isothermal amplification and its application. Addressing the core bottlenecks of the aforementioned existing technologies, this invention successfully constructs a quadruple RPA detection system for human herpesviruses for the first time. This is the first time globally that quadruple nucleic acid simultaneous amplification has been achieved in an RPA system, regardless of whether it targets the same class or different types of pathogens. Specifically, this is achieved through the following techniques.

[0005] In a first aspect, the present invention provides a human herpesvirus quadruple RPA detection system, comprising a group A of detection primers or a group B of detection primers; the group A of detection primers includes detection primer pairs for detecting HSV-1, HSV-2, VZV and EBV respectively, as shown in SEQ ID NO. 1-8 respectively; the group B of detection primers includes detection primer pairs for detecting HCMV, HHV-6, HHV-7 and HHV-8 respectively, as shown in SEQ ID NO. 9-16 respectively.

[0006] Furthermore, the human herpesvirus quadruple RPA detection system also includes plasmid standards for eight viruses: HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7, and HHV-8.

[0007] Furthermore, Group A includes HSV-1, HSV-2, VZV, and EBV, with primer concentrations of 30 μM, 20 μM, 7.5 μM, and 7.5 μM, respectively.

[0008] Furthermore, group B includes HCMV, HHV-6, HHV-7, and HHV-8, with primer concentrations of 10 μM, 9 μM, 10 μM, and 5 μM, respectively.

[0009] A second aspect of the present invention provides a method for detecting human herpesvirus quadruple RPA without the purpose of disease diagnosis and treatment, characterized in that RPA amplification is performed using the human herpesvirus quadruple RPA detection system described in any one of the above claims.

[0010] Furthermore, in the RPA amplification system, the final concentration ratio of the detection primer pairs for HSV-1, HSV-2, VZV, and EBV in the A group of detection primers is 12:3:3:8.

[0011] Furthermore, in the RPA amplification system, the final concentration ratio of the detection primer pairs for detecting HCMV, HHV-6, HHV-7, and HHV-8 in the B group of detection primers is 12:3:3:8.

[0012] Furthermore, the reaction temperature for RPA amplification is 33-43℃. Even further, the reaction temperature for RPA amplification is 41℃, and the reaction time is 15 min.

[0013] Furthermore, the RPA amplification reaction time using the detection primer combination A is 10-30 min, and the RPA amplification reaction time using the detection primer combination B is 15-30 min. Even further, the RPA amplification reaction time using the detection primer combination A is 15 min, and the RPA amplification reaction time using the detection primer combination B is 20 min.

[0014] Furthermore, the amount of activator used for RPA amplification is 1.5-4 μL. Even further, the amount of activator used is 2.5 μL.

[0015] In a third aspect, the present invention provides a quadruple RPA detection primer set for human herpesviruses, comprising either a group A primer set or a group B primer set; the group A primer set includes primer pairs for detecting HSV-1, HSV-2, VZV, and EBV, as shown in SEQ ID NO. 1-8 respectively; the group B primer set includes primer pairs for detecting HCMV, HHV-6, HHV-7, and HHV-8, as shown in SEQ ID NO. 9-16 respectively.

[0016] Compared with the prior art, the advantages of the present invention are:

[0017] 1. The quadruple RPA detection system provided by this invention can complete amplification in only 15-20 minutes, which saves 70% of the time compared to the 2 hours of the traditional PCR detection method, greatly improving the detection efficiency and meeting the needs of rapid clinical diagnosis.

[0018] 2. High detection sensitivity, with a detection limit of 1×10⁻⁶. 3 A count of 50 copies / μl (i.e., 20 copies / μl) can detect lower concentrations of the virus, which is helpful for early diagnosis and treatment.

[0019] 3. High specificity: Whether using a single virus positive template or a mixed template of four viruses, the corresponding virus can be accurately detected; when cross-testing the A and B systems, no amplification bands will appear, and the accuracy and reliability of the detection results are high.

[0020] 4. The system is highly portable, requiring only a constant-temperature metal bath for testing. It does not require complex equipment and is suitable for on-site testing scenarios such as biopharmaceutical plants and primary hospitals, providing greater convenience for virus detection. Attached Figure Description

[0021] Figure 1 The results of gel electrophoresis before and after RPA product purification are compared. The target fragment sizes of HSV-1, VZV, EBV, and HSV-2 are 453 bp, 326 bp, 265 bp, and 159 bp, respectively.

[0022] Figure 2 The results of preliminary exploratory research on constructing a group A quadruple RPA detection system are presented. In Figure A, the mixed primers are: HSV-1 (453 bp) + VZV (326 bp) + EBV (265 bp); the mixed template consists of positive templates for three viruses. In Figure B, the mixed primers are: HSV-1 (453 bp) + VZV (326 bp) + EBV (265 bp) + HSV-2 (117 bp); the mixed template consists of positive templates for four viruses.

[0023] Figure 3 The amplification effects of different kits in the Group A quadruple RPA detection system are shown. Specifically: Lane 1: Primer concentrations for HSV-1, VZV, EBV, and HSV-2 are all 200 nM; Lane 2: Primer concentrations for HSV-1, VZV, EBV, and HSV-2 are 500 nM, 200 nM, 500 nM, and 500 nM, respectively; Lane 3: Primer concentrations for HSV-1, VZV, EBV, and HSV-2 are 500 nM, 150 nM, 200 nM, and 200 nM, respectively; Lane 4: Primer concentrations for HSV-1, VZV, EBV, and HSV-2 are 500 nM, 100 nM, 200 nM, and 200 nM, respectively.

[0024] Figure 4 The results show the amplification results of single primers and mixed primers in the group B quadruple RPA detection system. The mixed primers are: HHV-8 (454 bp) + HHV-6 (334 bp) + HCMV (257 bp) + HHV-7 (114 bp).

[0025] Figure 5The results show the screening results for the Group B quadruple RPA detection system amplification kit. Specifically: Lane 1: Primer concentrations for HHV-8, HHV-6, HCMV, and HHV-7 were all 200 nM; Lane 2: Primer concentrations for HHV-8, HHV-6, HCMV, and HHV-7 were 150 nM, 200 nM, 200 nM, and 500 nM, respectively; Lane 3: Primer concentrations for HHV-8, HHV-6, HCMV, and HHV-7 were 200 nM, 200 nM, 200 nM, and 500 nM, respectively; Lane 4: Primer concentrations for HHV-8, HHV-6, HCMV, and HHV-7 were 100 nM, 200 nM, 200 nM, and 500 nM, respectively.

[0026] Figure 6 The results of sensitivity studies for four herpesviruses using the RPA detection systems in groups A and B are presented. The mixed primers in Figure A are: HSV-1 (453 bp) + VZV (326 bp) + EBV (265 bp) + HSV-2 (117 bp). The mixed primers in Figure B are: HHV-8 (454 bp) + HHV-6 (334 bp) + HCMV (257 bp) + HHV-7 (114 bp).

[0027] Figure 7 The results of the sensitivity study for detecting one herpesvirus in the RPA detection systems of groups A and B are presented. The mixed primers in the AD diagram are: HSV-1 (453 bp) + VZV (326 bp) + EBV (265 bp) + HSV-2 (117 bp). The mixed primers in the EH diagram are: HHV-8 (454 bp) + HHV-6 (334 bp) + HCMV (257 bp) + HHV-7 (114 bp).

[0028] Figure 8 The results show the specificity verification results of the quadruple RPA system. Figures A and C represent the specificity verification results of the quadruple RPA detection system in group A; Figures B and D represent the specificity verification results of the quadruple RPA detection system in group B.

[0029] Figure 9The primer ratios for the quadruple RPA detection system were optimized. In Figure A, the primer concentrations for lane 1 (HSV-1, VZV, EBV, and HSV-2) were 500 nM, 150 nM, 150 nM, and 400 nM, respectively; for lane 2 (HSV-1, VZV, EBV, and HSV-2), the primer concentrations were 540 nM, 200 nM, 150 nM, and 500 nM, respectively; for lane 3 (HSV-1, VZV, EBV, and HSV-2), the primer concentrations were 540 nM, 150 nM, 150 nM, and 400 nM, respectively; and for lane 4 (HSV-1, VZV, EBV, and HSV-2), the primer concentrations were 600 nM, 150 nM, 150 nM, and 400 nM, respectively.

[0030] In Figure B, the primer concentrations for lane 1 (HHV-8, HHV-6, HCMV, and HHV-7) are all 200 nM; for lane 2 (HHV-8, HHV-6, HCMV, and HHV-7), the primer concentrations are 150, 200, 200, and 200 nM, respectively; and for lane 3 (HHV-8, HHV-6, HCMV, and HHV-7), the primer concentrations are 100, 180, 200, and 200 nM, respectively.

[0031] Figure 10 Figure A shows the optimized primer volume for the quadruple RPA detection system. Figure B shows the optimized primer volume for the quadruple RPA detection system.

[0032] Figure 11 The figures show the optimized reaction temperature results for the quadruple RPA detection system. Figure A shows the optimized reaction temperature results for group A of the quadruple RPA detection system; Figure B shows the optimized reaction temperature results for group B of the quadruple RPA detection system.

[0033] Figure 12 Figure A shows the optimized reaction time results for the quadruple RPA detection system. Figure B shows the optimized reaction time results for the quadruple RPA detection system in group A.

[0034] Figure 13 The figures show the optimization results of the activator dosage in the quadruple RPA system. Figure A shows the optimized activator dosage for group A of the quadruple RPA detection system; Figure B shows the optimized activator dosage for group B of the quadruple RPA detection system.

[0035] Figure 14 This diagram illustrates the application of a quadruple RPA detection system in cells. The AC graph represents the detection results of the quadruple RPA system in group A, while the DF graph represents the detection results of the quadruple RPA system in group B. Detailed Implementation

[0036] 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.

[0037] 1. Experimental materials

[0038] In the specific embodiments provided in this invention, the *E. coli* strain DH5α and the pMD18-T vector were deposited and provided by our laboratory. All cell lines and live HSV-1, HSV-2, VZV, EBV, and HCMV viruses 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.

[0039] Table 1. Cell lines used in the experiment

[0040]

[0041]

[0042] 2. Main experimental reagents

[0043] The main reagents required for the experiment are shown in Table 2 below.

[0044] Table 2 Main reagents required for the experiment

[0045]

[0046] Example 1: Cell culture, resuscitation, passage, cryopreservation, and DNA extraction

[0047] 1. Cell Culture

[0048] This embodiment describes cell culture based on the standard experimental methods summarized by the China Center for Type Culture Collection (CCTCC).

[0049] 2. Cell resuscitation

[0050] The cell samples were thawed, centrifuged, resuspended and cultured, and finally the culture flasks were placed in a 37°C, 5% CO2 incubator.

[0051] 3. DNA extraction

[0052] (1) Cell line DNA extraction: Cell DNA was extracted using the blood / cell / tissue genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. The DNA concentration was determined in advance and diluted to 50 ng / μL, then stored at -20℃.

[0053] (2) Viral DNA extraction: HSV-1, HSV-2, VZV, EBV and HCMV preserved at the China Center for Type Culture Collection (CCTCC) were used to obtain viral DNA using the OMEGA Viral DNA Kit and stored at -20℃.

[0054] Example 2: Construction of a quadruple RPA detection system

[0055] 1. Design of Quadruple RPA Detection Primers

[0056] Addressing the high genotypic similarity among eight viruses within the same herpesvirus family, this invention establishes the core principle of primer design: "intraspecific conservation, significant interspecies differences, and gradient distribution of fragment sizes," overcoming the technical bottlenecks of cross-reactivity and detection confusion in conventional designs. To ensure the universality of primers for different strains of the same virus, this invention retrieves multiple reference sequences for each virus from the NCBI database (e.g., HSV-1 covers 10 strains including NC_001806.2, and HSV-2 covers 8 strains including NC_001798.2), and uses ClustalX for sequence alignment to screen regions with no base variation for more than 200 bp as candidate targets. For example, the 453 bp fragment of HSV-1 originates from the conserved region of the UL30 gene, which showed 99.2% sequence identity in 15 clinical isolates; the 326 bp fragment of VZV is located in the ORF62 gene, showing no base difference in 12 globally circulating strains, ensuring broad coverage of the primers for the same virus.

[0057] To address the issue of high sequence homology among viruses within the same family, the BLAST whole-genome alignment method was employed to calculate the sequence similarity of candidate target regions among eight viruses, rigorously screening fragments with similarity <60%. Specifically, the designed primer sequences were compared with the whole genomes of the other seven viruses using the Primer-BLAST tool to ensure that the primer binding regions exhibited continuous differences of ≥5 bases across species. For example, the 257 bp fragment of HCMV showed only 42% similarity to the corresponding region of HSV-1, and the 198 bp fragment of HHV-8 showed less than 38% homology to HHV-6, thus preventing cross-amplification at the sequence level.

[0058] Finally, the following genes were selected: HSV-1 (NC_001806.2), HSV-2 (NC_001798.2), VZV (NC_001348.1), EBV (NC_001847.1), HCMV (NC_006273.2), HHV-6 (NC_001664.4), HHV-7 (NC_001716.2), and HHV-8 (NC_009333.1), and a total of 50 primer pairs were designed.

[0059] 2. Primer screening

[0060] Through single RPA amplification and electrophoresis screening, 27 pairs of non-specific or inefficient primers were eliminated, and 33 pairs of highly specific primers were retained. For example, primers for HHV-7 were optimized in 3 rounds, and the 114 bp fragment with the highest amplification efficiency was selected to avoid cross-binding with the homologous region of HHV-6. Specifically, the primer pairs for the HSV-1 gene (NC_001806.2) are shown in SEQ ID NO. 1 and 2; the primer pairs for the HSV-2 gene (NC_001798.2) are shown in SEQ ID NO. 3 and 4; the primer pairs for VZV (NC_001348.1) are shown in SEQ ID NO. 5 and 6; the primer pairs for EBV (NC_001847.1) are shown in SEQ ID NO. 7 and 8; the primer pairs for HCMV (NC_006273.2) are shown in SEQ ID NO. 9 and 10; the primer pairs for HHV-6 (NC_001664.4) are shown in SEQ ID NO. 11 and 12; and the primer pairs for HHV-7 (NC_001716.2) are shown in SEQ ID NO. 12. Primer pairs for HHV-8 (NC_009333.1) are shown in NO.13 and 14, and are shown in SEQ ID NO.15 and 16.

[0061] 3. Construction of a quadruple RPA detection combination

[0062] The primer pairs for the eight HHVs ultimately selected were divided into groups A and B. During the grouping process, numerous combinations were experimented with. Regarding compatibility optimization for multiplex amplification, to avoid interference between primers, the amplified fragment sizes of the primers in both groups of quadruple RPA systems were kept significantly different to ensure clear differentiation and uniform distribution during electrophoresis, preventing small fragments from being masked. Specifically, group A detected HSV-1, HSV-2, VZV, and EBV, with corresponding amplified fragment sizes of 453 bp, 117 bp, 326 bp, and 265 bp, respectively; group B detected HCMV, HHV-6, HHV-7, and HHV-8, with amplified fragment sizes of 257 bp, 334 bp, 114 bp, and 454 bp, respectively. This gradient fragment design ensured that the amplified products of different viruses formed clearly distinguishable bands on the agarose gel, without overlap or masking.

[0063] The quadruple RPA detection system for Group A was established using the Xende Gene Basic Nucleic Acid Amplification Kit (see Table 3), with the four primer concentrations being 30 μM, 20 μM, 7.5 μM, and 7.5 μM, respectively. The quadruple RPA detection system for Group B was established using the TwistAmp® Basic kit (see Table 3), with the four primer concentrations being 10 μM, 9 μM, 10 μM, and 5 μM, respectively. Due to the complex composition of the quadruple RPA detection system, to better explore the conditions, we uniformly replaced the eight human herpesvirus positive templates with corresponding viral plasmid standards during the system establishment and optimization process.

[0064] Table 3. Quadruple RPA detection system for Group A and Group B

[0065]

[0066] During our experiment, we observed that some bands of the RPA amplification products shifted upwards after electrophoresis, especially the larger target fragments. We speculated that this might be due to the presence of complex components such as various enzymes in the product, which slowed down the migration rate during electrophoresis. To verify this hypothesis, we purified the amplification products and performed gel electrophoresis again. Figure 1 As shown in the figure, the target fragment size of HSV-1 is 453 bp; the target fragment size of VZV is 326 bp; the target fragment size of EBV is 265 bp; and the target fragment size of HSV-2 is 159 bp. It was found that the target band size of HSV-1 returned to normal.

[0067] (1) Establishment of Group A's quadruple RPA detection system

[0068] After determining that the Group A quadruple RPA detection system could detect HSV-1, HSV-2, VZV, and EBV, we initially chose the TwistAmp® Basic kit to establish the quadruple RPA detection system, but multiple experiments failed. Therefore, based on the results of preliminary experiments (RPA experiments with individual templates and primers), we mixed primers for HSV-1, VZV, and EBV in equal proportions and added corresponding positive templates for amplification, thus establishing a triple RPA detection system. The results are as follows: Figure 2 As shown in Figure A, all specific target bands are consistent with expectations, indicating that the system can simultaneously detect these three viruses. Based on this, we further validated multiple pairs of HSV-2 primers to construct a quadruple RPA detection system. After continuous screening, we finally determined the specific primers for HSV-2, which amplify a target fragment of 117 bp. These primers were added to the original triple RPA detection system, and amplification was performed using four single viral plasmids and a mixed template, respectively. The results are shown in Figure A. Figure 2As shown in Figure B, although the primers are highly specific, they cannot amplify four viruses simultaneously, and the quadruple RPA detection system has not yet been successfully constructed.

[0069] Since we were unable to amplify all four fragments simultaneously despite multiple adjustments to the primer concentrations, we considered switching to an isothermal amplification kit. To compare amplification results, we set up four experimental groups, using the XGMA Basic Nucleic Acid Amplification Kit and the TwistAmp® Basic kit for simultaneous amplification. The results are as follows: Figure 3 As shown in the figure, it can be observed that among the four experimental groups amplified by the TwistAmp® Basic kit, only the VZV and EBV target fragments were successfully amplified, while the target bands of HSV-1 and HSV-2 were hardly amplified; however, all four experimental groups using the kit produced by Xianda Gene Technology Co., Ltd. could amplify four corresponding target fragments, but the brightness of the four bands was not uniform. Among them, the four bands in experimental group 2 were more obvious, and the target band of EBV was the brightest.

[0070] Therefore, we decided to use the XNGene basic nucleic acid amplification kit to further adjust the primer concentration to optimize the group A quadruple RPA detection system.

[0071] (2) Establishment of Group B's quadruple RPA detection system

[0072] Similarly, after determining the Group B quadruple RPA detection system for HHV-8, HHV-6, HCMV, and HHV-7, we constructed the Group B quadruple RPA detection system using the TwistAmp® Basic kit and finally determined the primers for HHV-8, HHV-6, HCMV, and HHV-7, with target fragment lengths of 454 bp, 334 bp, 257 bp, and 114 bp, respectively. Amplification was then performed by adding the corresponding positive template with a single primer, such as... Figure 4 A. Preliminary assessment indicates that the primers have good specificity.

[0073] Subsequently, we mixed the four primers in equal proportions to form a primer set, constructed the group B quadruple RPA detection system, and analyzed the amplification products by gel electrophoresis. The results are as follows: Figure 4 As shown in B, specific bands can be observed in the mixed primer set, whether only one virus or all four viruses are present, proving that the system can simultaneously detect four viruses: HHV-8, HHV-6, HCMV, and HHV-7. The quadruple RPA detection system of group B has been successfully constructed.

[0074] To screen the optimal isothermal amplification kit for the Group B quadruple RPA detection system, we compared the amplification effects of the TwistAmp® Basic kit and the Citronix Basic Nucleic Acid Amplification Kit. The results are as follows: Figure 5 As shown. Unlike the Group A quadruple RPA detection system, the Group B quadruple RPA detection system, when amplified using the SindaGene Isothermal Amplification Basic Kit, could not simultaneously amplify all four fragments; it could only amplify EBV and HHV-7 bands with poor results. In contrast, although the four experimental groups amplified by the TwistAmp® Basic kit could simultaneously detect four viruses. Therefore, in subsequent experiments, we decided to continue using the TwistAmp® Basic kit to further optimize the Group B quadruple RPA detection system.

[0075] Example 3: Preparation of Virus Standards

[0076] 1. Amplification of the target fragment

[0077] (1) Design of primers for amplification of the target fragment

[0078] Based on literature review and NCBI comparison, and using the genes of the aforementioned eight herpesviruses, the sequences shown in SEQ ID NO. 17-24 were selected as the target fragments, and corresponding amplification primers were designed. The primer design principles were: primer length 25-30 bp, annealing temperature 55-65℃, and GC content 35%-50%. The designed primers were then compared and analyzed using the Primer Blast module of NCBI. Furthermore, the HHV-7 plasmid and all primers designed in this experiment were synthesized by Wuhan Tianyi Huiyuan Co., Ltd.

[0079] (2) Amplification and gel recovery of the target fragment

[0080] The target fragment was amplified using the PCR reaction system in Table 5 and the PCR reaction procedure in Table 6. The amplified product was recovered using the ordinary agar gel DNA recovery kit from Tiangen Biotech (Beijing) Co., Ltd., and sequencing confirmed its accuracy.

[0081] Table 5 PCR reaction system

[0082]

[0083]

[0084] Table 6 PCR reaction procedure

[0085]

[0086] 2. Connection Conversion

[0087] Double-digest the accurately sequenced target fragment and the pMD18-T plasmid. Perform agarose gel electrophoresis on the plasmid digestion products, and store the recovered products at 4°C. Purify the target fragment digestion products using a purification kit from Tiangen Biotech (Beijing) Co., Ltd., and set aside. Take the recovered pMD18-T plasmid vector and the purified target fragment, and ligate them to obtain the ligation product (i.e., the recombinant expression vector containing the target fragment), and set aside.

[0088] The ligation product was transformed into competent cells, cultured, and the colony growth was observed. When colonies were clearly visible and no satellite colonies had formed, a single colony was identified and inoculated into LB broth containing ampicillin for expansion. Finally, amplification was performed, and the cells were identified by agarose gel electrophoresis. Samples matching the expected results were sequenced. Correct sequencing results confirmed successful transformation. Successfully transformed DH5α cells were then preserved in glycerol and stored at -80°C for later use.

[0089] 3. Plasmid extraction

[0090] Take the successfully transformed DH5α, extract the plasmid according to the plasmid mini-prep kit operation manual of Tiangen Biotech (Beijing) Co., Ltd., sequence it, and after confirming that it is correct, use it for later use.

[0091] 4. Preparation of Standards

[0092] Remove the extracted plasmid from the refrigerator and measure its concentration using a Nanodrop 2000c nucleic acid concentration analyzer. Calculate the molecular weight and concentration of the plasmid. Divide the plasmid from 10... 10 Gradually dilute to 10 copies / μL. 1 Copies / μL, with a total of 10 concentration gradients. Prepare 10 μL of plasmid with a concentration of 1×10⁻⁶ copies / μL. 10 A standard mixture of (copies / μL).

[0093] Example 4: Sensitivity Study of a Quadruple RPA System

[0094] Each viral plasmid was diluted to a concentration of 1 × 10⁻⁶. 7 1×10 6 1×10 5 1×10 4 1×10 3 1×10 2 1×10 1 and 1×10 0 Copies / μL were used to investigate the sensitivity of the quadruple RPA system in groups A and B for detecting mixed and single viruses.

[0095] To investigate the sensitivity of simultaneous detection of four viruses, 1 μL of each of the four plasmids from each gradient, totaling 4 μL, was used as template for amplification. To investigate the sensitivity of the quadruple RPA system for detecting a single virus, 1 μL of the plasmid corresponding to each virus gradient was used as template in a mixed primer set for amplification. After the reaction, 5 μL of 10× Loading Buffer was added and mixed thoroughly by pipetting. 10 μL was then taken for agarose gel electrophoresis to observe the results. The results are as follows: Figure 6 As shown, it can also be clearly observed that when both groups A and B of the quadruple RPA detection systems simultaneously detect four human herpesviruses, the lowest detection limit can reach 1×10⁻⁶. 3 20 copies / μL (50 μL system).

[0096] We also investigated the detection sensitivity of single human herpesviruses in detection systems A and B. Taking HSV-1 as an example, we added 1 μL of HSV-1 plasmids at different concentration gradients to each reaction system. The procedure for other human herpesviruses was similar, and all were amplified under optimal conditions. The agarose gel electrophoresis results are shown below. Figure 7 As shown, the AD graph represents the sensitivity results of the quadruple RPA detection system in group A for detecting a single herpesvirus, and the EG graph represents the sensitivity results when the viral plasmid copy number is ≥1×10⁻⁶. 3 When the target band is less than or equal to 1×10, it can be observed, but when it is less than or equal to 1×10, the target band is less visible. 2 At that time, the target band could not be detected. Therefore, the detection limit for a single virus in both quadruple RPA detection systems can reach 1×10⁻⁶. 3 Copy number (50 μL system) is 20 copies / μL.

[0097] Example 5: Specificity Validation of the Quadruple RPA System

[0098] To ensure the reliability of the test results, we verified the specificity of systems A and B. During the establishment and optimization of the quadruple RPA detection system, all herpesvirus positive templates used were constructed plasmid standards.

[0099] 1. Validation of the detection performance of the quadruple RPA system in complex samples

[0100] To verify the effectiveness of the quadruple RPA detection system in complex samples, for group A, we replaced the viral positive templates for HSV-1, VZV, and HSV-2 with DNA extracted from their respective live viruses, and replaced the viral positive template for EBV with DNA from B95-8 cells. For group B, we replaced the viral positive templates with DNA from HHV-7 plasmid, HCMV virus, and DNA from BCBL-1 (HHV-8) and HUV-EC-C (HHV-6) cells. Single viral positive templates and mixed templates were added separately, and amplification was performed under optimal conditions.

[0101] The results are as follows Figure 8 As shown in Figures A and B, when the quadruple RPA detection system detects only one virus, it amplifies only one corresponding specific target fragment; when four viruses are present at the same time, the system can also detect them simultaneously, thus verifying the specificity of the quadruple RPA detection system.

[0102] 2. To explore the effectiveness of cross-validation of the two quadruple RPA systems, A and B.

[0103] To investigate whether non-specific amplification occurred during cross-detection of the two quadruple RPA systems (A and B), the A detection system was used to detect four positive templates from group B; conversely, the B detection system was used to detect four positive templates from group A. Specifically, the A system was used to detect HHV-7 plasmid, HCMV viral DNA, and DNA from BCBL-1 (HHV-8) and HUV-EC-C (HHV-6) cells; the B detection system was used to detect HSV-1, HSV-2, and VZV viral DNA, as well as B95-8 cell DNA (EBV). A negative control was included. Electrophoresis was performed and the amplification results were analyzed.

[0104] The results are as follows Figure 8 As shown in Figures C and D, all results were negative. This further confirms the specificity of the two quadruple RPA detection systems, thus ensuring the accuracy and reliability of the test results.

[0105] Example 6: Optimization of a quadruple RPA reaction detection system

[0106] First, the total volume of the mixed primers in the quadruple RPA system was fixed at 4 μL. Four different ratios were set up for group A quadruple RPA reaction detection systems, and three different ratios were set up for group B quadruple RPA reaction detection systems. After determining the ratios, eight experiments were conducted to optimize the total volume of the mixed primers. Next, the reaction time was fixed at 20 min, and the reaction temperature was optimized by setting six temperature gradients: 35, 37, 39, 41, 43, and 45 °C. Then, the reaction time was optimized again, with the temperature fixed at 41 °C, and amplification was performed for 5, 10, 15, 20, 25, and 30 mins. Finally, the amount of activator was optimized, and the optimal reaction conditions were determined by observing the gel electrophoresis results after the reaction.

[0107] 1. Primer concentration optimization

[0108] Previous experiments have determined that the Group A quadruple RPA detection system uses the Xende Gene Basic Nucleic Acid Amplification Kit, while the Group B quadruple RPA detection system uses the TwistAmp® Basic kit. To improve detection efficiency, we optimized the primer concentrations.

[0109] Specifically, the total primer volume was first fixed at 4 μL, and the ratio of the four primer pairs was optimized. For the group A quadruple RPA detection system, we set up four different primer ratios for amplification. Results Figure 9 As shown in Figure A, the fourth experimental group exhibited the best amplification effect. At this point, the primer ratio of HSV-1, VZV, EBV, and HSV-2 in the system was 10:3:3:4. Therefore, this ratio was determined to be the optimal primer ratio for the quadruple RPA detection system in group A.

[0110] For the group B quadruple RPA detection system, we set up different primer ratios for amplification. The results are as follows: Figure 9 As shown in Figure B, the amplification efficiency of the four fragments in the three experimental groups was relatively uniform after adjustment, with the third experimental group showing the best results. At this point, the primer ratio of HHV-8, HHV-6, HCMV, and HHV-7 in the system was 5:9:10:10. Therefore, this ratio was determined to be the optimal primer ratio for the quadruple RPA detection system in Group B.

[0111] Next, we mixed the primers in equal volumes according to the optimal ratio to prepare primer sets, optimized the total volume of primer sets, and set up a total of 8 experimental groups for amplification. The experimental results of the quadruple RPA detection system in group A are as follows: Figure 10A. It was found that when the total volume of the mixed primers increased to 7 μL, the target band of VZV showed non-specific amplification. Comparison revealed that the amplification effect was optimal at a total primer volume of 6 μL, which was determined as the optimal primer amount. At this point, the final primer concentrations of HSV-1, VZV, EBV, and HSV-2 in the detection system of group A were 900, 225, 225, and 600 nM, respectively. The results of the quadruple RPA detection system of group B are as follows... Figure 10 In group B, it can be observed that the brightness of the target band increases with the increase of primer dosage, and it is clear that the target band is sufficiently clear when the total primer dosage is 8 μL, thus determining it as the optimal primer dosage. At this point, the final primer concentrations of HHV-8, HHV-6, HCMV, and HHV-7 in the quadruple RPA detection system of group B are 200, 360, 400, and 400 nM, respectively.

[0112] 2. Optimization of reaction temperature

[0113] To investigate the optimal reaction temperature for the two quadruple RPA detection systems (groups A and B), we set up six temperature gradients for amplification: 33, 35, 37, 39, 41, and 43℃. The results for group A are as follows... Figure 11 As shown in Figure A, the results indicate that four bands can be amplified within this temperature range, and it is clearly observed that the amplification effect is optimal at 41℃. Further increases in temperature are detrimental to band amplification. Therefore, we ultimately selected 41℃ as the optimal reaction temperature. The results for group B are as follows... Figure 11 As shown in Figure B, the results indicate that within this temperature range, the target fragment gradually becomes clearer as the temperature increases; however, the bands become blurred at ≥43℃. Therefore, we determined that the optimal reaction temperature for the quadruple RPA detection system in group B is also 41℃.

[0114] 3. Optimization of reaction time

[0115] To determine the optimal reaction time for the quadruple RPA detection system, we set six time gradients: 5, 10, 15, 20, 25, and 30 min; electrophoresis analysis was performed immediately after the reaction. The results for group A are shown below. Figure 12 As shown in Figure A, the target band was not observable at an amplification time of 5 min. With increasing amplification time, the target band gradually became clearer; at a reaction time of 15 min, the target band was sufficiently clear. As the time continued to increase, the specific band of VZV gradually diffused. Therefore, 15 min was determined to be the optimal reaction time for the system. The results for Group B are shown below. Figure 12 As shown in Figure B, when the amplification time is ≤10 min, the target band does not amplify significantly; as the reaction time increases, the band brightness increases. When the reaction time is 20 min, the band brightness is sufficient. Therefore, the optimal reaction time for the detection system in group B is determined to be 20 min.

[0116] 4. Optimization of activator dosage

[0117] The activator is crucial for initiating the RPA reaction. To investigate the optimal amount of activator in a quadruple RPA detection system, we set up six experimental groups to optimize it, and then performed gel electrophoresis on the products. The results for group A are shown below. Figure 13 As shown in Figure A, the target band is relatively faint when the activator dosage is 1.5 μL. When the activator dosage is 2 μL, the specific target band is sufficiently clear. As the activator dosage continues to increase, the target band gradually becomes diffuse. Therefore, the optimal activator dosage for the quadruple RPA detection system in group A is 2 μL. The results for group B are shown below. Figure 13 As shown in B, when the amount of activator is in the range of 1.5-4 μL, the system can amplify four specific target fragments. Careful observation revealed that when the amount of activator added is 2.5 μL, the target band is sufficiently clear. When the amount of activator is increased to 3.5 μL, the target band begins to become blurred. Therefore, the optimal amount of activator for the quadruple RPA detection system in group B is determined to be 2.5 μL.

[0118] In summary, this embodiment optimized the conditions of the two quadruple RPA detection systems (Group A and Group B) in terms of primer concentration, reaction temperature, reaction time, and activator dosage. The final determined conditions were as follows: In Group A, the final primer concentrations for HSV-1, VZV, EBV, and HSV-2 were 900 nM, 225 nM, 225 nM, and 600 nM, respectively; the optimal reaction temperature was 41℃; the optimal reaction time was 15 min; and the optimal activator dosage was 2 μL. In Group B, the final primer concentrations for HHV-8, HHV-6, HCMV, and HHV-7 were 200 nM, 360 nM, 400 nM, and 400 nM, respectively; the optimal reaction temperature was 41℃; the optimal reaction time was 20 min; and the optimal activator dosage was 2.5 μL.

[0119] Application Example 1: Application of the Quadruple RPA Detection System in Cells

[0120] Multiplex RPA detection systems are important for the regular screening of cell banks. To verify the detection efficacy of group A and group B detection systems for human herpesvirus in cells, more than 30 cell lines listed in Table 1 were detected using both systems. The detection results are as follows: Figure 14 As shown, one cell line was EBV positive (ARH-77 cells); the rest were negative, a result consistent with cell literature. This confirms the high efficiency and reliability of the quadruple RPA detection system (Groups A and B) of this invention in actual cell sample testing, and also demonstrates its broad application prospects in cell bank screening and virus detection.

[0121] 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. A human herpesvirus quadruple RPA detection system, characterized in that, It includes either Group A or Group B detection primer combinations; Group A detection primer combinations include primer pairs for detecting HSV-1, HSV-2, VZV, and EBV respectively, and Group B detection primer combinations include primer pairs for detecting HCMV, HHV-6, HHV-7, and HHV-8 respectively. The nucleotide sequences of the detection primer pairs for HSV-1 are shown in SEQ ID NO.1 and SEQ ID NO.2; The nucleotide sequences of the detection primer pairs for HSV-2 are shown in SEQ ID NO.3 and SEQ ID NO.42; The nucleotide sequences of the detection primer pairs for VZV detection are shown in SEQ ID NO.5 and SEQ ID NO.6; The nucleotide sequences of the detection primer pairs for EBV detection are shown in SEQ ID NO.7 and SEQ ID NO.8; The nucleotide sequences of the detection primer pairs for HCMV detection are shown in SEQ ID NO.9 and SEQ ID NO.10; The nucleotide sequences of the detection primer pairs for HHV-6 are shown in SEQ ID NO.11 and SEQ ID NO.12; The nucleotide sequences of the detection primer pairs for HHV-7 are shown in SEQ ID NO.13 and SEQ ID NO.14; The nucleotide sequences of the detection primer pairs for HHV-8 are shown in SEQ ID NO.15 and SEQ ID NO.

16.

2. The human herpesvirus quadruple RPA detection system according to claim 1, characterized in that, It also includes plasmid standards for eight viruses: HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7, and HHV-8.

3. The human herpesvirus quadruple RPA detection system according to claim 1, characterized in that, Group A includes HSV-1, HSV-2, VZV, and EBV, with primer concentrations of 30 μM, 20 μM, 7.5 μM, and 7.5 μM, respectively; Group B includes HCMV, HHV-6, HHV-7, and HHV-8, with primer concentrations of 10 μM, 9 μM, 10 μM, and 5 μM, respectively.

4. A fourfold RPA detection method for human herpesviruses not intended for disease diagnosis and treatment, characterized in that, RPA amplification was performed using the human herpesvirus quadruple RPA detection system according to any one of claims 1-3.

5. The human herpesvirus quadruple RPA detection method according to claim 4, not for disease diagnosis and treatment, is characterized in that, In the RPA amplification system, the final concentration ratio of the detection primer pairs for HSV-1, HSV-2, VZV and EBV in the A group of detection primers is 12:3:3:

8. In the RPA amplification system, the final concentration ratio of the detection primer pairs for detecting HCMV, HHV-6, HHV-7, and HHV-8 in the B group of detection primers is 12:3:3:

8.

6. The human herpesvirus quadruple RPA detection method according to claim 4, not for disease diagnosis and treatment, is characterized in that, The reaction temperature for RPA amplification is 33-43℃; Furthermore, the reaction temperature for RPA amplification is 41°C, and the reaction time is 15 min.

7. The human herpesvirus quadruple RPA detection method according to claim 4, not for disease diagnosis and treatment, is characterized in that, The RPA amplification reaction time using the detection primer combination of group A is 10-30 min, and the RPA amplification reaction time using the detection primer combination of group B is 15-30 min. Furthermore, the RPA amplification reaction time using the detection primer combination of group A is 15 min, and the RPA amplification reaction time using the detection primer combination of group B is 20 min.

8. The human herpesvirus quadruple RPA detection method according to claim 4, not for the purpose of disease diagnosis and treatment, is characterized in that, The amount of activator used for RPA amplification is 1.5-4 μL; Furthermore, the amount of activator used for RPA amplification is 2.5 μL.

9. The human herpesvirus quadruple RPA detection method according to claim 4, not for disease diagnosis and treatment, is characterized in that, The A-group detection primer combination is used in combination with the Xende Gene Basic Nucleic Acid Amplification Kit; the B-group detection primer combination is used in combination with the TwistAmp® Basic kit.

10. A quadruple RPA detection primer set for human herpesvirus, characterized in that, The detection primer combination can be either Group A or Group B; Group A includes primer pairs for detecting HSV-1, HSV-2, VZV, and EBV respectively, and Group B includes primer pairs for detecting HCMV, HHV-6, HHV-7, and HHV-8 respectively. The nucleotide sequences of the detection primer pairs for HSV-1 are shown in SEQ ID NO.1 and SEQ ID NO.2; The nucleotide sequences of the detection primer pairs for HSV-2 are shown in SEQ ID NO.3 and SEQ ID NO.4; The nucleotide sequences of the detection primer pairs for VZV detection are shown in SEQ ID NO.5 and SEQ ID NO.6; The nucleotide sequences of the detection primer pairs for EBV detection are shown in SEQ ID NO.7 and SEQ ID NO.8; The nucleotide sequences of the detection primer pairs for HCMV detection are shown in SEQ ID NO.9 and SEQ ID NO.10; The nucleotide sequences of the detection primer pairs for HHV-6 are shown in SEQ ID NO.11 and SEQ ID NO.12; The nucleotide sequences of the detection primer pairs for HHV-7 are shown in SEQ ID NO.13 and SEQ ID NO.14; The nucleotide sequences of the detection primer pairs for HHV-8 are shown in SEQ ID NO.15 and SEQ ID NO.16.

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