Kit for multi-typing detection of human herpes viruses HHV-1 to HHV-8 and use method

Abstract

The invention discloses a kit for multi-typing detection of human herpes viruses HHV-1 to HHV-8, and the kit comprises a group of nucleic acid combination which comprises 9 pairs of RPA (recombinase polymerase amplification) primers which are respectively and specifically combined with target genes of HHV-1 to HHV-8 and an EC (exogenous internal control), wherein each pair of primers comprises a forward primer and a reverse primer; wherein the 5'ends of the reverse primers of all the 9 pairs of primers are marked with biotin; the 5'ends of forward primers of the HHV-1, the HHV-4 and the HHV-7 are marked with first haptens; the 5'end of the forward primer of the HHV-2, the HHV-5 and the HHV-8 is marked with a second hapten; the positive primers of the HHV-3 and the HHV-6 are marked with a third hapten at the 5'end; a fourth hapten is marked at the 5'end of the forward primer of the EC; the invention relates to an RPA (recombinase polymerase amplification) reaction buffer solution which comprises the following components in concentration: 40-60 mM of Tris-HCl, 80-140 mM of potassium acetate, 10-20 mM of magnesium acetate, 3-8% (w / v) of polyethylene glycol and 0.5-2.0 M of betaine. And a plurality of lateral chromatography test strips.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to a kit for detecting multiple types of human herpes virus HHV-1 to HHV-8 and a use method thereof. BACKGROUND

[0002] HHV (human herpes virus) is a kind of double-stranded DNA virus widely spread in human population, which has the characteristics of universality, latency and recurrence. According to its biological characteristics, HHV family can be divided into three subfamilies of α, β and γ, which contains at least eight viruses closely related to human diseases, namely HSV-1 (type 1 herpes simplex virus), HSV-2 (type 2 herpes simplex virus), VZV (varicella-zoster virus, i.e. HHV-3), EBV (EB virus, i.e. HHV-4), HCMV (human cytomegalovirus, i.e. HHV-5), HHV-6 (human herpes virus 6), HHV-7 (human herpes virus 7) and KSHV (Kaposi's sarcoma-associated herpes virus, i.e. HHV-8).

[0003] Different subtypes of HHV can cause a variety of diseases, from common cold sores, genital herpes, chickenpox, to severe viral encephalitis, mononucleosis, and even closely related to the occurrence and development of various malignant tumors (such as nasopharyngeal carcinoma, Burkitt's lymphoma, Kaposi's sarcoma). Especially in immunocompromised populations, such as AIDS patients, organ transplant recipients or cancer chemotherapy patients, latent infection of HHV is activated, which can lead to fatal systemic disseminated infection. Because the clinical manifestations of diseases caused by different subtypes of HHV are cross and atypical, and their treatment regimens (such as the selection and dosage of antiviral drugs) are specific, therefore, rapid and accurate typing diagnosis of HHV has crucial clinical significance for guiding clinical medication, evaluating prognosis and controlling virus transmission.

[0004] Currently, the "gold standard" method for HHV detection in clinical practice mainly relies on laboratory techniques, such as virus culture, serological antibody detection and PCR (polymerase chain reaction) based nucleic acid detection. Virus culture takes too long (several days to several weeks), and has strict requirements for samples and laboratory conditions; serological antibody detection has a "window period", which cannot distinguish between current infection and past infection, and may produce false negative results in immunocompromised patients. Although qPCR (real-time fluorescent quantitative PCR) technology has high sensitivity and specificity, its operation process is complex, requiring precise temperature control circulator and professional experimenters, which is difficult to meet the POCT (Point-of-Care Testing) needs in primary medical institutions, community clinics or home self-testing scenarios.

[0005] To overcome the above limitations, in recent years, a variety of isothermal nucleic acid amplification techniques have emerged, among which, RPA (Recombinase Polymerase Amplification) technology has shown great potential in the field of POCT due to its mild reaction conditions (usually 37-42℃ constant temperature), fast speed (amplification completed within 15-30 minutes) and low requirement for equipment. The combination of RPA technology and LFD (Lateral Flow Dipstick) can realize the visual interpretation of the amplification product, further simplifying the operation, so that the whole detection process does not require any large instrument.

[0006] However, the inventors found through in-depth research on the prior art that the application of the RPA-LFD technology platform to high-throughput typing detection of all eight HHVs still faces the following technical bottlenecks that need to be solved urgently: First, the implementation of high-throughput multiplex RPA amplification is extremely difficult. Second, the existing lateral flow test strip cannot meet the clear presentation of high-throughput results. Third, there is a lack of integrated solutions suitable for HHV multiplex typing detection. An ideal POCT product should be able to handle convenient non-invasive samples such as saliva. However, the existing sample pretreatment methods are often tedious or incompatible with downstream amplification systems. Currently, there is no technology that can seamlessly integrate efficient saliva sample pretreatment, robust 8-way RPA amplification, and 8-channel result interpretation without crosstalk, forming a complete and smooth Sample-to-Answer (from sample to result) POCT system.

[0007] In summary, the prior art has obvious gaps and technical obstacles in realizing rapid, simple and high-throughput typing detection of all eight human herpes viruses. There is an urgent need in the art for an innovative technical solution to overcome the problems of multiplex amplification interference and multi-channel result presentation, thereby providing a truly effective tool for the instant diagnosis of HHV-related diseases. SUMMARY

[0008] The present application provides a kit and use method for multiplex detection of human herpes viruses HHV-1 to HHV-8, which is used to overcome the inherent defects of recombinase polymerase amplification technology as an instant diagnosis method in the multiplex detection and typing of human herpes viruses.

[0009] In a first aspect of the present application, a kit for multiplex typing of human herpesviruses HHV-1 to HHV-8 is provided, which comprises: a set of nucleic acid combinations comprising 9 pairs of RPA (recombinase polymerase amplification) primers specifically binding to target genes of HHV-1 to HHV-8 and one EC (exogenous internal control) respectively, wherein each pair of primers comprises a forward primer and a reverse primer; wherein the reverse primers of all 9 pairs of primers are labeled with biotin at the 5' end; the forward primers of HHV-1, HHV-4, and HHV-7 are labeled with a first hapten at the 5' end; the forward primers of HHV-2, HHV-5, and HHV-8 are labeled with a second hapten at the 5' end; the forward primers of HHV-3 and HHV-6 are labeled with a third hapten at the 5' end; the forward primer of EC is labeled with a fourth hapten at the 5' end; a RPA reaction buffer, the component concentrations of which comprise 40-60 mM Tris-HCl, 80-140 mM potassium acetate, 10-20 mM magnesium acetate, 3-8% (w / v) polyethylene glycol, and 0.5-2.0 M betaine; a plurality of lateral flow test strips, the conjugate pad of the lateral flow test strip is pre-coated with streptavidin-labeled nanoparticles, and a first detection line for capturing the first hapten, a second detection line for capturing the second hapten, a third detection line for capturing the third hapten, a fourth detection line for capturing the fourth hapten, and a control line (C line) for capturing biotin are arranged on the detection zone of the lateral flow test strip.

[0010] In a second aspect of the present application, a method for multiplex typing of human herpesviruses 1 to 8 in vitro is provided, which comprises: sample pretreatment: taking 200 μL of saliva sample, adding 200 μL of sample lysate containing EC template, shaking and mixing, centrifuging briefly, and taking 5 μL of lysate supernatant as sample nucleic acid; RPA amplification: adding 5 μL of pretreated sample nucleic acid to 45 μL of RPA reaction system, and performing amplification reaction at a constant temperature of 25-42°C to obtain amplification product within 10-20 minutes; lateral flow detection: taking the lateral flow test strip, dropping 50 μL of RPA reaction product onto the test strip I, II, and III, placing the test strip, and visually reading the results; result interpretation: checking the C line of the test strip I, II, and III and the detection line of EC, which must all develop color; if any C line or EC detection line does not develop color, the test strip result is invalid, and a new test strip must be used to retest the original amplification product; according to the color development of one or more of the detection lines, the type of one or more human herpesviruses present in the sample to be tested is determined.

[0011] In a third aspect, the present application provides use of the kit according to any one of claims 1-7 in the preparation of a diagnostic product for in vitro multiplex diagnosis of human herpes virus 1-8 infection.

[0012] The one or more technical solutions provided in the present application have at least the following technical effects or advantages:

[0013] The embodiments of the present application successfully solve the long-standing technical problems in the field of high-throughput multiplex RPA detection by designing an innovative and synergistic reaction system, and provide a HHV multiplex detection scheme far superior to the existing technical level, with ultra-high throughput, ultra-high speed and ultra-high sensitivity, which has significant progress and outstanding substantial characteristics.

[0014] The above description is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application, and in order to make the above and other purposes, characteristics and advantages of the present application more apparent and easy to understand, the following specific embodiments of the present application are described. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative labor.

[0016] Figure 1 The detection results of the analog HHV-2 / HHV-6 co-infection sample in the prior art. DETAILED DESCRIPTION

[0017] After introducing the basic principles of the present application, the technical solutions in the present application will be described clearly and completely with reference to the drawings. Obviously, the described embodiments are only some of the embodiments of the present application, not all embodiments of the present application. It should be understood that the present application is not limited by the example embodiments described here. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application. In addition, it should be noted that, for convenience of description, only parts related to the present application are shown in the drawings, not all.

[0018] Embodiment one

[0019] Design and synthesis of primers

[0020] 1.1 Primer design and screening The present application preliminarily designed multiple pairs of RPA primers according to the highly conserved and specific gene sequences of each type of human herpes virus 1-8 (HHV-1-8) included in the GenBank database, as well as a piece of artificially designed non-natural sequence as an exogenous internal control (EC). The primer design followed the basic principles of RPA technology, with a length of 30-35 bp and a GC content of 40-60%. Subsequently, through BLAST comparison analysis, the primer pairs with potential cross-reactivity with the human genome or other common pathogens were excluded to ensure the high specificity of the primers.

[0021] 1.2 Primer synthesis and modification The optimal primer pairs screened were synthesized and PAGE purified by a commercial company (Shenguo Bioengineering (Shanghai) Co., Ltd.). According to the "grouped hapten labeling" detection strategy of the present application, the primers were chemically modified. The detailed information is shown in Table 1:

[0022] Table 1: Primer information used in the present application

[0023]

[0024]

[0025] The target sequence of the EC is an artificially synthesized DNA fragment, as shown in SEQ ID NO: 19, which has no homology with known biological sequences.

[0026] The sequence listing is as follows:

[0027] SEQ ID NO: 1 CCGACAGCACCGACGATGAACGCCGACGTGAT

[0028] SEQ ID NO: 2 CGTCCAGTCGTTCTCGTCGATGGCGTAGTTGA

[0029] SEQ ID NO: 3 GACATCTACGCGGGGCTCAACTTCAACATCAC

[0030] SEQ ID NO: 4 GGTGTAGATGGCGGCGTACAGGTCGTTGATCT

[0031] SEQ ID NO: 5 AATGGACGCCGATTACACCAACGAGTATGACA

[0032] SEQ ID NO: 6 GTTGTCGTCGTCGTCGTTGATCTTGCCGTCGT

[0033] SEQ ID NO: 7 TCTGGCGAGGTTGGTGTTTACTGCTGAGGAGA

[0034] SEQ ID NO: 8 GTCGTCCTCATCTCTGTTCCTCTAGTCTTGGC

[0035] SEQ ID NO: 9 AGTTTACGACGTGTCCAACGAGCGCATCGTCA

[0036] SEQ ID NO: 10 GTAGATCACGATCACGTCGTCGTCGATCACGA

[0037] SEQ ID NO: 11 TGTATTTGGTGTCTGTGGCTACGACTACGTCT

[0038] SEQ ID NO: 12 GACATCGGCTGTACCATCGTCGTTGACGTTGA

[0039] SEQ ID NO: 13 AAGGACAAGAGCGGCAACTACACCGTCAACAA

[0040] SEQ ID NO: 14 GTCGTCGTCGTCGTTGATGACGTCGTCGTCGT

[0041] SEQ ID NO: 15 AGCCATAGGCTCCACGGCCATAAATGACACAT

[0042] SEQ ID NO: 16 GTCGTCGTCGTCGTCGTCGTCGTCGTCGTCGTC

[0043] SEQ ID NO: 17 GCAACAGTCGACGTTAAATTCGGCGTTACCGG

[0044] SEQ ID NO: 18 CACTCAAGTAAATTCGTTGACGATCCGGTCAA

[0045] SEQ ID NO: 19 GCAACAGTCGACGTTAAATTCGGCGTTACCGGATCGTCAACGAATTTACTTGAGTGTTGACGATCCGGTCAACGAATTTACTTGAGTG

[0046] Example Two

[0047] Preparation of each component of the kit

[0048] 2.1 Preparation of sample lysis solution The sample lysis solution was prepared with the final concentrations of 20 mM Tris-HCl (pH 8.0), 1 M guanidine hydrochloride, 0.5% (v / v) Triton X-100, 200 μg / mL proteinase K, 50 mM DTT, and 2 mM EDTA. The EC template (SEQ ID NO: 19) was diluted and added to the lysis solution with a final concentration of 100 copies / μL. After aliquoting, it was stored at -20 °C.

[0049] 2.2 Preparation of RPA reaction system The RPA reaction system was provided in two parts for storage and use: (1) RPA reaction dry powder pellet: the RPA basic enzyme components (including T4 uvsX recombinase, T4 uvsY protein, Bsu DNA polymerase), dNTPs (200 μM each), phosphocreatine (50 mM), and 9 pairs of primers (the final concentrations of HHV-1 to 8 primers were 240 nM each, and the final concentration of EC primer was 80 nM) were mixed and freeze-dried to form a dry powder pellet that was easy to reconstitute and placed at the bottom of a PCR reaction tube. (2) RPA reaction buffer: the reaction buffer was accurately prepared to contain 50 mM Tris-HCl (pH 8.3), 100 mM potassium acetate, 14 mM magnesium acetate, 5% (w / v) polyethylene glycol, 1.0 M betaine, and 50 mM phosphocreatine. This buffer was a reconstitution solution before use.

[0050] 2.3 Preparation of lateral chromatographic test strip (1) Preparation of SA-gold labeled probe: streptavidin solution was mixed with colloidal gold nanoparticles (average particle size 40 nm) prepared by the trisodium citrate reduction method under optimized pH conditions for covalent coupling. After centrifugal purification, it was resuspended with a buffer containing 1% BSA to form an SA-gold labeled probe, which was uniformly sprayed on a glass fiber membrane and dried at 37 °C for 2 hours to serve as a binding pad. (2) Coating and assembly of NC membrane: on the nitrocellulose membrane (NC membrane), the capture antibody and protein were accurately coated using a membrane drawing gold spraying instrument. The coating solution concentration was 1 mg / mL, and the coating amount was 1 μL / cm.

[0051] Test strip I (for HHV-1 / 2 / 3): From the sample end to the absorbent pad end, anti-FAM antibody (T1 line), anti-DIG antibody (T2 line), anti-Dansyl antibody (T3 line), and biotinylated bovine serum albumin (Biotin-BSA) as a control line (C line) were drawn in order. The line spacing was 5 mm.

[0052] Test strip II (for HHV-4 / 5 / 6): Anti-FAM antibody (T4 line), anti-DIG antibody (T5 line), anti-Dansyl antibody (T6 line) and Biotin-BSA (C line) are drawn in turn.

[0053] Test strip III (for HHV-7 / 8 / EC): Anti-FAM antibody (T7 line), anti-DIG antibody (T8 line), anti-FITC antibody (T9 line, for EC) and Biotin-BSA (C line) are drawn in turn. The treated sample pad, conjugate pad, NC membrane and absorbent pad are laminated and pasted on the PVC base plate in turn, cut into 3 mm wide test strips, and sealed and packaged in a dry environment.

[0054] Example Three

[0055] Verification of synergistic effect of multiplex RPA reaction system

[0056] To verify the synergistic effect of magnesium acetate, PEG and betaine in the present application, this example quantitatively investigates the influence of different component combinations on the amplification speed in a complex system containing all 9 pairs of primers, taking HHV-1 as a model. Method: The reaction system containing 10 3 copies of HHV-1 template is subjected to RPA amplification at 39°C, and the reaction tube is taken out every 2 minutes, and its product is applied to test strip I, and the shortest time for the appearance of the naked eye visible T1 line is recorded. Each condition is repeated 3 times. Results: The quantitative influence of different buffer components on the amplification speed of 9 multiplex RPA is shown in Table 2 below.

[0057] Table 2: Quantitative influence of different buffer components on the amplification speed of 9 multiplex RPA

[0058]

[0059] Conclusion: The data clearly show that under the complex conditions of 9 multiplex amplification, the basic buffer (Group A) cannot effectively amplify. The addition of PEG (Group B) or betaine (Group C) alone can start the reaction, but the speed is slower. When the three are used in combination at the optimized concentration of the present application (Group D), the amplification speed is increased by about 34% and 42% compared with Groups B and C, respectively, showing a significant, non-additive synergistic effect.

[0060] Example Four

[0061] Performance comparison between 9 multiplex detection and single detection

[0062] To prove that the present application solves the technical problem of performance decline caused by multiplex amplification and achieves the unexpected technical effect of "faster multiplex detection than single detection", we carried out a strict head-to-head comparison. Method:

[0063] Single detection group: only one pair of primers for one HHV was added in the RPA system.

[0064] 9plex detection group: all 9 pairs of primers were added in the RPA system.

[0065] 4.1 Sensitivity (LOD) determination: HHV-1, HHV-2, HHV-5, HHV-8 plasmid standards containing 1000, 200, 100, 50, 20, 10, 0 copies / reaction were detected, each concentration was repeated 8 times, and the lowest concentration corresponding to at least 95% detection rate (i.e. at least 7 positive out of 8 times) was taken as the LOD. 4.2 Speed determination: the above templates containing 100 copies / reaction were detected, and the time of positive signal appearing was recorded.

[0066] Table 3: Performance comparison data of 9plex detection and single detection

[0067]

[0068] Example Five

[0069] Specific detection

[0070] Objective: To verify whether the 9plex RPA-LFA kit of the present application will produce cross-reaction to non-target types of HHV viruses, i.e. to test its ability to distinguish different HHV types.

[0071] Methods: 8 groups of reaction systems were prepared. In each group, high concentration (1 x 10 6 copies / reaction) of plasmid DNA standard of HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7 or HHV-8 was added as template. All reactions used the complete 9plex RPA reaction system (containing all 9 pairs of primers and synergistic buffer) prepared in Example 2. The amplification reaction was carried out at 39°C for 15 minutes. The amplification products were applied to test strips I, II, III, and the results were read after standing for 10 minutes.

[0072] Result reading standard: observed with naked eye, "+" indicates the appearance of clear red bands, "-" indicates the absence of any bands. Each experiment was repeated 3 times, and the results must be consistent.

[0073] Results: The detailed results of cross-reaction test are shown in Table 4 below.

[0074] Table 4: Results of HHV-1 to HHV-8 cross-reaction test

[0075]

[0076] Conclusion: As shown in Table 4, when using high concentration of single HHV template for detection, only its corresponding detection line (T line) showed positive result, all other 7 HHV detection lines were negative. All reactions' internal control line (T9) and quality control line (C) showed normal color development. This strongly proved that the 9 pairs of primers in the kit of the present application were highly specific, in the complex 9-reaction system, each target sequence could still be accurately identified, and there was no cross-reaction between each other.

[0077] 5.2 Specific detection

[0078] Objective: To verify whether the kit of the present application will have cross-reaction with other common pathogens that may exist in clinical samples (especially saliva samples).

[0079] Methods: Multiple groups of reaction systems were prepared. In each group, high concentration (1 x 10 7 copies / reaction or equivalent concentration) of the following pathogen nucleic acid extracts were added:

[0080] Influenza A virus

[0081] Influenza B virus

[0082] Adenovirus

[0083] Respiratory Syncytial Virus (RSV)

[0084] Human Coronavirus OC43

[0085] Streptococcus pneumoniae

[0086] Staphylococcus aureus

[0087] All reactions used the complete 9-reaction RPA reaction system prepared in Example 2. The amplification reaction was carried out at 39°C for 15 minutes. The amplification product was applied to test strips I, II, III, and the results were read after 10 minutes of standing.

[0088] Results: The detailed results of specific detection are shown in Table 5 below.

[0089] Table 5: Specific detection results with other common pathogens

[0090]

[0091]

[0092] Conclusion: As shown in Table 5, no positive signal appeared in all HHV detection lines (T1-T8) when the kit of the present application was used to detect a variety of common non-target pathogens at high concentrations. This fully demonstrates that the kit of the present application has extremely high analytical specificity, and the detection results will not be interfered by other common pathogens that may coexist in clinical samples.

[0093] Conclusion: Through the detailed data verification of Examples 5.1 and 5.2, the 9-reagent RPA-LFA kit of the present application can accurately distinguish 8 different types of HHV, and effectively exclude cross-reactions with other common pathogens, ensuring the high accuracy and reliability of the detection results.

[0094] Example Six

[0095] Detection process of simulated clinical samples

[0096] Method: Simulate the clinical HHV-2 and HHV-6 co-infection saliva sample. Add 10 3 copies of HHV-2 and HHV-6 plasmid standard to 200 μL of saliva of healthy people confirmed to be free of HHV viruses, respectively.

[0097] Sample processing (5 minutes): Take 200 μL of the above simulated sample, add 200 μL of sample lysate containing EC template, vortex for 15 seconds, and stand at room temperature for 1 minute.

[0098] RPA amplification (15 minutes): Take 5 μL of lysate supernatant, add it to the reaction tube containing RPA reaction dry powder ball, add 45 μL of RPA synergistic buffer, tightly cover the tube, mix well, and incubate in a 39°C constant temperature metal bath for 15 minutes.

[0099] Chromatographic detection (5 minutes): Add 50 μL of amplification product to the sample pad of test strip I, II, III, respectively, and read the results after standing at room temperature for 5 minutes. Results: As shown in Table 6. Figure 1

[0100] Conclusion: The results clearly show that the T2 line of test strip I and the T6 line of test strip II are strongly positive, and all C lines and the T9 line of EC are positive, which is completely consistent with the expected results. It indicates that the kit of the present application can accurately and reliably detect and distinguish a variety of HHV infections in clinical samples, and the whole process can be completed within 25 minutes, which has extremely strong practicability.

[0101] ​Through these specific embodiments, it can be clearly seen that the present application is a kit for detecting multiple types of human herpes virus 1 to 8 (HHV-1 to HHV-8), which, through its unique synergistic reaction system containing magnesium acetate, polyethylene glycol and betaine, as well as the ingenious multiple hapten labeling and lateral chromatographic detection design, not only simplifies the detection process of multiple pathogens, but also performs well in sensitivity and specificity, and is suitable for the rapid diagnosis needs of clinical and public health.

[0102] In summary, the present application successfully solves the technical problems of strong inhibition effect, low sensitivity and slow speed of existing RPA technology in high multiplicity (9-fold) amplification, and achieves the revolutionary and unexpected technical effect of "faster multiple detection than single detection". The present application provides a brand-new technical solution integrating ultra-high throughput, ultra-high speed, high sensitivity, high specificity and easy operation, which far exceeds the existing technical level. Therefore, the present application effectively overcomes various shortcomings of the prior art and has high industrial utilization value.

[0103] In another embodiment of the present application, the detection component can also include eight independent lateral chromatographic test strips, each of which is provided with a detection line for a specific HHV subtype amplification product. In this case, the RPA amplification reaction product can be aliquoted into eight parts and added dropwise to the eight independent test strips for detection. Although this method may be more cumbersome in operation than using a single array test strip, it can also achieve the technical purpose of 8-fold typing detection as required by the present application, and therefore also falls within the protection scope of the present application.

[0104] Although the present application has been described in conjunction with specific features and embodiments thereof, it is obvious that various modifications and combinations can be made thereto without departing from the spirit and scope of the present application. Accordingly, the present specification and drawings are merely illustrative of the present application, and any and all modifications, variations, combinations or equivalents that are within the scope of the present application are intended to be embraced by the present application. Obviously, those skilled in the art can make various modifications and changes to the present application without departing from the scope of the present application. Thus, if these modifications and changes of the present application belong to the scope of the present application and its equivalent technology, the present application is intended to include these modifications and changes.

Claims

1. A kit for the multiplex typing of human herpes viruses HHV-1 to HHV-8, characterized in that, The kit comprises: (a) a set of nucleic acid combinations comprising 9 pairs of recombination enzyme polymerase amplification primers respectively specifically binding to target genes of HHV-1 to HHV-8 and one exogenous internal control, wherein each pair of primers comprises a forward primer and a reverse primer; wherein the reverse primers of all 9 pairs of primers are labeled with biotin at the 5' end; the forward primers of HHV-1, HHV-4, HHV-7 are labeled with a first hapten at the 5' end; the forward primers of HHV-2, HHV-5, HHV-8 are labeled with a second hapten at the 5' end; the forward primers of HHV-3, HHV-6 are labeled with a third hapten at the 5' end; the forward primer of EC is labeled with a fourth hapten at the 5' end; (b) an RPA reaction buffer, the concentration of the components of which comprises 40-60mM Tris-HCl, 80-140mM potassium acetate, 10-20mM magnesium acetate, 3-8% w / v polyethylene glycol, and 0.5-2.0M betaine; (c) a plurality of lateral flow test strips, the conjugate pad of the lateral flow test strip is pre-coated with streptavidin-labeled nanoparticles, and a detection zone is provided on the lateral flow test strip, which comprises: a first detection line for capturing the first hapten; a second detection line for capturing the second hapten; a third detection line for capturing the third hapten; a fourth detection line for capturing the fourth hapten; a control line for capturing biotin.

2. The kit of claim 1, wherein: the first hapten is fluorescein; the second hapten is digoxin; the third hapten is dansyl chloride; the fourth hapten is fluorescein isothiocyanate.

3. The kit of claim 1 or 2, wherein: the kit comprises three lateral flow test strips I, II and III for detecting HHV amplification products in different groups, respectively, wherein: lateral flow test strip I is provided with first, second and third detection lines corresponding to HHV-1, HHV-2 and HHV-3, respectively; lateral flow test strip II is provided with first, second and third detection lines corresponding to HHV-4, HHV-5 and HHV-6, respectively; lateral flow test strip III is provided with first and second detection lines corresponding to HHV-7 and HHV-8, respectively, and a fourth detection line corresponding to EC.

4. The kit of claim 1, wherein: the RPA reaction buffer comprises 30-60mM phosphocreatine, and the concentration of each component is matched so that in a reaction system containing all 9 pairs of primers, the time required for a positive result is shortened by at least 10% compared to the corresponding reaction system containing only any single primer pair, while the detection sensitivity for each target is not less than 100 copies / reaction.

5. The kit of claim 1, wherein: The RPA reaction buffer comprises 50 mM Tris-HCl with a pH of 8.3, 100 mM potassium acetate, 14 mM magnesium acetate, 5% w / v polyethylene glycol, 1.0 M betaine, and 50 mM phosphocreatine.

6. The kit of claim 1, wherein: The nucleotide sequences of the 9 pairs of RPA primers are shown in SEQ ID NOs: 1-18, respectively, and the exogenous internal control detection target sequence is an artificially synthesized DNA with the following sequence: SEQ ID NO: 1 CCGACAGCACCGACGATGAACGCCGACGTGAT SEQ ID NO: 2 CGTCCAGTCGTTCTCGTCGATGGCGTAGTTGA SEQ ID NO: 3 GACATCTACGCGGGGCTCAACTTCAACATCAC SEQ ID NO: 4 GGTGTAGATGGCGGCGTACAGGTCGTTGATCT SEQ ID NO: 5 AATGGACGCCGATTACACCAACGAGTATGACA SEQ ID NO: 6 GTTGTCGTCGTCGTCGTTGATCTTGCCGTCGT SEQ ID NO: 7 TCTGGCGAGGTTGGTGTTTACTGCTGAGGAGA SEQ ID NO: 8 GTCGTCCTCATCTCTGTTCCTCTAGTCTTGGC SEQ ID NO: 9 AGTTTACGACGTGTCCAACGAGCGCATCGTCA SEQ ID NO: 10 GTAGATCACGATCACGTCGTCGTCGATCACGA SEQ ID NO: 11 TGTATTTGGTGTCTGTGGCTACGACTACGTCT SEQ ID NO: 12 GACATCGGCTGTACCATCGTCGTTGACGTTGA SEQ ID NO: 13 AAGGACAAGAGCGGCAACTACACCGTCAACAA SEQ ID NO: 14 GTCGTCGTCGTCGTTGATGACGTCGTCGTCGT SEQ ID NO: 15 AGCCATAGGCTCCACGGCCATAAATGACACAT SEQ ID NO: 16 GTCGTCGTCGTCGTCGTCGTCGTCGTCGTCGTC SEQ ID NO: 17 GCAACAGTCGACGTTAAATTCGGCGTTACCGG SEQ ID NO: 18 CACTCAAGTAAATTCGTTGACGATCCGGTCAA SEQ ID NO: 1 CCGACAGCACCGACGATGAACGCCGACGTGAT SEQ ID NO: 2 CGTCCAGTCGTTCTCGTCGATGGCGTAGTTGA SEQ ID NO: 3 GACATCTACGCGGGGCTCAACTTCAACATCAC SEQ ID NO: 4 GGTGTAGATGGCGGCGTACAGGTCGTTGATCT SEQ ID NO: 5 AATGGACGCCGATTACACCAACGAGTATGACA SEQ ID NO: 6 GTTGTCGTCGTCGTCGTTGATCTTGCCGTCGT SEQ ID NO: 7 TCTGGCGAGGTTGGTGTTTACTGCTGAGGAGA SEQ ID NO: 8 GTCGTCCTCATCTCTGTTCCTCTAGTCTTGGC SEQ ID NO: 9 AGTTTACGACGTGTCCAACGAGCGCATCGTCA SEQ ID NO: 10 GTAGATCACGATCACGTCGTCGTCGATCACGA SEQ ID NO: 11 TGTATTTGGTGTCTGTGGCTACGACTACGTCT SEQ ID NO: 12 GACATCGGCTGTACCATCGTCGTTGACGTTGA SEQ ID NO: 13 AAGGACAAGAGCGGCAACTACACCGTCAACAA SEQ ID NO: 14 GTCGTCGTCGTCGTTGATGACGTCGTCGTCGT SEQ ID NO: 15 AGCCATAGGCTCCACGGCCATAAATGACACAT SEQ ID NO: 16 GTCGTCGTCGTCGTCGTCGTCGTCGTCGTCGTC SEQ ID NO: 17 GCAACAGTCGACGTTAAATTCGGCGTTACCGG SEQ ID NO: 18 CACTCAAGTAAATTCGTTGACGATCCGGTCAA ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ SEQ ID NO: 19 GCAACAGTCGACGTTAAATTCGGCGTTACCGGATCGTCAACGAATTTACTTGAGTGTTGACGATCCGGTCAACGAATTTACTTGAGTG.

7. The kit of claim 1, wherein: the sample lysate comprises 10-50 mM Tris-HCl, 0.5-2.0% Triton X-100, 0.5-2 M guanidine hydrochloride, and 100-500 μg / mL proteinase K, 1-5 mM EDTA, 20-100 mM DTT, and 50-200 copies / μL of exogenous internal control template.

8. A method for multiplex typing of human herpes virus types 1 to 8 in vitro, characterized in that, The method comprises the following steps: (a) sample pretreatment: take 200 μL of saliva sample, add 200 μL of sample lysate containing EC template, shake and mix, centrifuge briefly, and take 5 μL of lysate supernatant as sample nucleic acid; (b) RPA amplification: add 5 μL of treated sample nucleic acid to 45 μL of RPA reaction system, and perform amplification reaction at a constant temperature of 25-42°C to obtain amplification product within 10-20 minutes; (c) lateral flow test: take out the lateral flow test strip, drop 50 μL of RPA reaction product on test strips I, II, and III, lay the test strip flat, wait and visually interpret the results; (d) result interpretation: check the C line and EC detection line of test strips I, II, and III, which must all develop color; if any one of the C line or the EC detection line does not develop color, the test strip result is invalid, and a new test strip must be used to retest the original amplification product; according to the color development of one or more of the detection lines, the type of one or more human herpes viruses present in the sample to be tested is determined.

9. Use of the kit of any one of claims 1-7 in the preparation of a diagnostic product for in vitro multiplex diagnosis of human herpes virus type 1-8 infection.

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