Primer probe combinations and kits for detecting orthopoxvirus and detecting and typing monkeypox virus

Abstract

The present application relates to a primer-probe combination and a kit for detecting orthopoxvirus and detecting and typing monkeypox virus, wherein the primer-probe combination comprises at least one of the following (1) to (2): (1) a primer pair for orthopoxvirus with a sequence as shown in SEQ ID NO: 3 and SEQ ID NO: 5, and a probe for orthopoxvirus with a sequence as shown in SEQ ID NO: 7; (2) a primer pair for monkeypox virus with a sequence as shown in SEQ ID NO: 13 and SEQ ID NO: 16, a probe for type I monkeypox virus with a sequence as shown in SEQ ID NO: 17, and a probe for type II monkeypox virus with a sequence as shown in SEQ ID NO: 18. The primers can be used to detect orthopoxvirus and different types of monkeypox virus in the same reaction system, with good detection specificity, high sensitivity and higher accuracy.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a primer-probe combination and a kit for detecting orthopoxvirus and monkeypox virus and typing. Background Art

[0002] Orthopoxviruses (family Poxviridae, genus Orthopoxvirus) are a group of large enveloped viruses containing a covalently closed double-stranded DNA genome of approximately 200 kbp. In addition to Variola virus, this genus contains more than 10 known important animal and zoonotic viruses, such as Monkeypox virus (MPXV), Cowpox virus, Raccoonpox virus, Mousepox virus, etc. Symptoms of human infection with orthopoxviruses range from mild skin lesions to fatal systemic diseases. For example, smallpox produces a generalized rash that progresses from papules to blisters to pustules, resulting in a mortality rate of more than 30% in unvaccinated people. Although the World Health Organization (WHO) announced in 1980 that smallpox virus had been eradicated, it is still considered a potential biological threat because it can be naturally transmitted through droplets and aerosols, has an extremely high infection rate and mortality rate, and due to the cessation of smallpox vaccination, human immunity to smallpox has declined. In addition to smallpox, several other members of the genus can cause human infection, including monkeypox, cowpox, and other zoonotic rodent-borne orthopoxviruses.

[0003] After the smallpox virus was declared eradicated, MPXV became the most serious orthopoxvirus. The virus was first isolated from a Congolese boy in 1970. From 1997 to 2020, the virus broke out many times in Africa, including the People's Republic of the Congo and Nigeria, infecting more than 5,000 people and killing more than 200 people. In May 2022, monkeypox cases were reported in non-endemic countries. In the following three months, the monkeypox epidemic continued and began to spread globally. On July 23, 2022, the World Health Organization (WHO) officially announced that the global monkeypox outbreak was an international public health emergency. The epidemic has involved more than 100 countries.

[0004] MPXV has two phylogenetic branches: the Central African (Congo Basin) evolutionary branch and the West African evolutionary branch. The Congo Basin branch has historically caused more severe disease, with a mortality rate of up to 10.6%, while the West African branch is about 3%, but the West African branch is considered to be more transmissible. On August 12, 2022, WHO referred to the former Central African (Congo Basin) evolutionary branch as the first evolutionary branch (I) and the former West African evolutionary branch as the second evolutionary branch (II). Given the differences in clinical symptoms, mortality rates and transmission efficiency between clades I and II, their accurate typing is of great significance to the spread and control of the disease.

[0005] At present, monkeypox virus mainly relies on laboratory testing, and the detection methods include virus isolation and culture, electron microscopy, immunohistochemistry, antigen or antibody detection, nucleic acid detection, etc. Among them, virus isolation and identification is the classic method for diagnosing monkeypox virus, but it requires high laboratory safety level and a long detection cycle. The specificity of antigen or antibody serological methods is not high. The most common method is PCR detection, but the existing PCR detection often has low sensitivity and weak specificity, and can only detect monkeypox virus, and cannot detect other orthopoxviruses at the same time, nor can it type the detected monkeypox virus, which is not conducive to differential diagnosis and special prevention and control of the epidemic. Summary of the invention

[0006] Based on this, it is necessary to provide a primer probe combination, a kit and a detection method that can simultaneously detect monkeypox virus and other orthopoxviruses, and can type the I and II evolutionary branches of monkeypox virus, and have the advantages of high sensitivity and high specificity.

[0007] The specific technical solutions are as follows:

[0008] A primer-probe combination, the primer-probe combination comprising at least one of the following (1) to (2):

[0009] (1) a primer pair for orthopoxvirus having a sequence as shown in SEQ ID NO: 3 and SEQ ID NO: 5, and a probe for orthopoxvirus having a sequence as shown in SEQ ID NO: 7;

[0010] (2) A primer pair for monkeypox virus with sequences as shown in SEQ ID NO: 13 and SEQ ID NO: 16, a probe for monkeypox virus type I with sequence as shown in SEQ ID NO: 17, and a probe for monkeypox virus type II with sequence as shown in SEQ ID NO: 18.

[0011] A virus detection kit, comprising any one of the primer-probe combinations.

[0012] A method for detecting orthopoxvirus and / or detecting monkeypox virus and distinguishing between monkeypox virus type I and type II for non-diagnostic and therapeutic purposes, the method comprising the following steps:

[0013] Use any one of the primer-probe combinations or any one of the virus detection kits to perform fluorescent quantitative PCR analysis on the nucleic acid in the sample to obtain analysis results.

[0014] Compared with the prior art, this application has the following beneficial effects:

[0015] The present application provides a primer-probe combination that can simultaneously detect monkeypox virus and other orthopoxviruses, and can type monkeypox virus I and II clades. On this basis, a corresponding detection kit is also provided. The primers and the kit can be used to detect orthopoxviruses and different types of monkeypox viruses in the same reaction system, with good detection specificity, high sensitivity and more accuracy, while shortening the detection time to achieve the purpose of rapid detection, which is of great significance for the treatment of monkeypox virus-infected people and the control of the epidemic. Furthermore, the kit can also be stored at room temperature, has good stability, and has strong repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of the primer and probe combination screening of ORP; wherein the horizontal axis is cycle, the numerical range of the horizontal axis is 0-40, and the vertical axis is ΔRn, the numerical range of the vertical axis is 0-600000;

[0017] Figure 2 The primer and probe combination screening results of IC; wherein the horizontal axis is cycle, the numerical range of the horizontal axis is 0-40, and the vertical axis is ΔRn, the numerical range of the vertical axis is 0-450000;

[0018] Figure 3 The results of the primer and probe combination screening for MPV;

[0019] Figure 4 This is a finished product display picture of the freeze-dried reagent of sample 1;

[0020] Figure 5 This is a finished product display picture of sample 2 freeze-dried reagent;

[0021] Figure 6 This is a finished product display picture of sample 3 freeze-dried reagent;

[0022] Figure 7 This is a finished product display picture of sample 4 freeze-dried reagent;

[0023] Figure 8 This is a finished product display picture of sample 5 freeze-dried reagent;

[0024] Fig. 9 The test results for cowpox virus and vaccinia virus;

[0025] Fig.10 This is the test result of monkeypox virus type I;

[0026] Fig.11 This is the test result of monkeypox virus type II;

[0027] Fig.12 The results are for the detection of monkeypox virus type I and type II;

[0028] Fig.13 This is a negative result of the detection of a non-orthopoxvirus sample in Example 5. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0031] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0032] Primer probe combination

[0033] One embodiment of the present application provides a primer-probe combination, which includes a primer pair for orthopoxviruses with sequences such as SEQ ID NO: 3 and SEQ ID NO: 5, and a probe for orthopoxviruses with sequences such as SEQ ID NO: 7. Using the primer-probe combination, orthopoxviruses can be quickly detected, with the advantages of high sensitivity, low cost, and short detection time.

[0034] In a specific example, the orthopoxvirus includes one or more of monkeypox virus, mousepox virus, cowpox virus, and vaccinia virus.

[0035] In a specific example, both ends of the probe are modified with a fluorescent group and a quenching group, respectively. Optionally, the fluorescent group is selected from one of FAM, HEX, VIC, CY5, ROX, Texsa Red and Quasar 705, and optionally, the quenching group is selected from one of BHQ1, BHQ2, BHQ3, TAMRA and MGB. It should be noted that in the primer-probe combination, the fluorescent groups of different probes are different from each other and do not interfere with each other.

[0036] In a specific example, the 5' end of the orthopoxvirus probe is modified with a fluorescent group ROX, and the 3' end is modified with a quencher group BQ2.

[0037] One embodiment of the present application further provides a primer-probe combination, which includes a primer pair of monkeypox virus as shown in SEQ ID NO: 13 and SEQ ID NO: 16, a probe of monkeypox virus type I as shown in SEQ ID NO: 17, and a probe of monkeypox virus type II as shown in SEQ ID NO: 18. Using the primer-probe combination, monkeypox virus can be quickly detected in one tube, and monkeypox virus type I and type II can be distinguished at the same time.

[0038] In the present application, monkeypox virus type I represents the first evolutionary branch (I) of monkeypox virus; and monkeypox virus type II represents the second evolutionary branch (II) of monkeypox virus.

[0039] In one specific example, the probes of monkeypox virus type I and type II are modified with different fluorescent groups. In one specific example, the 5' end of the probe of monkeypox virus type I is modified with the fluorescent group FAM, and the 5' end of the probe of monkeypox virus type II is modified with the fluorescent group VIC. In another specific example, the 5' end of the probe of monkeypox virus type I is modified with the fluorescent group VIC, and the 5' end of the probe of monkeypox virus type II is modified with the fluorescent group FAM.

[0040] The detection of monkeypox virus type I and monkeypox virus type II uses a pair of primers in common. The probe 3' is modified with a quenching group MGB, and the binding stability of the probe to the template is greatly increased, which can fully type monkeypox virus type I and monkeypox virus type II when they share a pair of primers.

[0041] One embodiment of the present application also provides a primer-probe combination, which can be used to quickly detect orthopoxvirus and monkeypox virus in one tube. When the test result is monkeypox virus, it can simultaneously distinguish between monkeypox virus type I and type II, so that different viruses can be treated differently, thereby improving the efficiency of treatment and prevention.

[0042] Specifically, the primer-probe combination includes the following groups (1) to (2):

[0043] (1) a primer pair for orthopoxvirus having a sequence as shown in SEQ ID NO: 3 and SEQ ID NO: 5, and a probe for orthopoxvirus having a sequence as shown in SEQ ID NO: 7; (2) a primer pair for monkeypoxvirus having a sequence as shown in SEQ ID NO: 13 and SEQ ID NO: 16, a probe for monkeypoxvirus type I having a sequence as shown in SEQ ID NO: 17, and a probe for monkeypoxvirus type II having a sequence as shown in SEQ ID NO: 18.

[0044] In a specific example, the probe of orthopoxvirus and the probe of monkeypoxvirus type I and type II are modified with different fluorescent groups; the probe of monkeypoxvirus type I and type II are modified with different fluorescent groups.

[0045] In a specific example, any of the above primer-probe combinations may also include a primer pair and a probe for an internal reference gene. Optionally, a primer pair for an internal reference gene having a sequence as shown in SEQ ID NO: 8 and SEQ ID NO: 10, and a probe for an internal reference gene having a sequence as shown in SEQ ID NO: 12.

[0046] In a specific example, the fluorescent groups of the probe of the internal reference gene, the probe of the orthopoxvirus, and the probe of monkeypox virus type I and type II are all different.

[0047] In a specific example, in the probe of the internal reference gene, the 5' end is modified with a fluorescent group CY5, and the 3' end is modified with a quenching group BQ2.

[0048] Uses of primer-probe combinations

[0049] One embodiment of the present application provides the use of any of the above primer-probe combinations in the preparation of products for detecting orthopoxviruses, and / or detecting monkeypox viruses and distinguishing between monkeypox virus types I and II.

[0050] Reagent test kit

[0051] One embodiment of the present application provides a virus detection kit capable of detecting orthopoxvirus and / or detecting and distinguishing monkeypox virus type I and type II. The kit includes reagents for nucleic acid amplification, and the reagents for nucleic acid amplification include the primer-probe combination described in any one of claims 1 to 5.

[0052] In a specific example, the reagents used for nucleic acid amplification also include one or more of Taq DNA polymerase, UNG enzyme, Tris-HCl, MgCl2, KCl and dNTP.

[0053] In a specific example, the reagent used for nucleic acid amplification is a lyophilized powder reagent or a mixed solution reagent.

[0054] In a specific example, the reagent used for nucleic acid amplification is a lyophilized powder reagent, and the reagent used for nucleic acid amplification also includes a lyophilized excipient. Optionally, the lyophilized excipient includes one or more of trehalose, mannitol, bovine serum albumin, Tween80, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), tert-butyl alcohol, lactose and sodium carboxymethyl cellulose.

[0055] Among them, lactose and trehalose are oligosaccharides that function as cryoprotectants during the freezing process and as dehydration protectants during the drying and dehydration process. Mannitol will crystallize during slow freezing, thus providing a support structure for the active substance, and mannitol will not react with the active component. BSA is an enzyme stabilizer that prevents enzyme decomposition and nonspecific adsorption. Tert-butyl alcohol has a high freezing point and a high vapor pressure, forming needle-shaped crystals, which can change the solute crystallization mode and facilitate sublimation. PVP, PEG and sodium carboxymethyl cellulose are polymer protective agents that are also very effective cryoprotectants during the low-temperature storage and freeze-drying of biological materials. They are preferentially precipitated during the freezing process, have certain surface activity, increase the viscosity of the solution, and significantly increase the glass transition temperature. Through the synergistic effect of trehalose and sodium carboxymethyl cellulose, the freeze-dried reagent can be stored at 37°C for more than 6 months with good stability. Even after the high temperature tolerance of the freeze-dried reagent is enhanced to 56°C, it can still be stored for 6 months, significantly improving the stability of the freeze-dried reagent. The invention solves the problems that the existing monkeypox PCR detection reagent needs to be frozen and cannot be stored at room temperature, which is not conducive to the storage, transportation and rapid detection of the reagent.

[0056] Specifically, the lyophilized powder reagent contains the following components at working concentrations:

[0057] 0.5U / reaction~2U / reaction Taq DNA polymerase, 0.1U / reaction~0.5U / reaction UNG enzyme, 10mM~80mMTris-HCl, 1.0mM~5.0mM MgCl2, 10mM~100mM KCl, 0.1μM~1μM dNTPs, 2%w / v~5%w / v trehalose, 2%w / v~5%w / v mannitol, 1%w / v~3%w / v bovine serum albumin, 0.1%v / v~0.6%v / v Tween80, 0.5%w / v~2%w / v PEG20000, 0.2%w / v~0.5%w / v PVP40000, 0.1% v / v-2% v / v tert-butyl alcohol, 1% w / v-3% w / v lactose, 1% w / v-3% w / v sodium carboxymethyl cellulose, 50 nM-200 nM of each primer and 50 nM-200 nM of each probe.

[0058] Specifically, the reconstitution solution is ultrapure water.

[0059] Specifically, the positive quality control product is a pseudovirus that encapsulates the monkeypox virus gene, and the negative quality control product is gDNA extracted from a human cell line.

[0060] In a specific example, the freeze-drying process used for the freeze-dried reagent includes the steps of pre-freezing, primary drying and desorption drying.

[0061] Specifically, the freeze-drying process includes the following steps:

[0062] Pre-freezing: at -50℃~-55℃, freeze-drying for 3h~4h; primary drying: at -40℃~-45℃, freeze-drying for 10h~12h, vacuum degree is 8Pa~12Pa; analytical drying: at 20℃~25℃, freeze-drying for 4h~5h, vacuum degree is 8Pa~12Pa.

[0063] Using the above reagent components and freeze-drying process of the present application, a room temperature storage kit can be prepared. The room temperature storage kit can be stored in an environment of 37° C. for more than 6 months with good stability.

[0064] Detection Methods

[0065] A method for detecting orthopoxvirus for non-diagnostic purposes, and / or detecting monkeypox virus and distinguishing between monkeypox virus type I and type II, the method comprising the following steps:

[0066] Use any one of the primer-probe combinations or the kit to perform fluorescent quantitative PCR analysis on the nucleic acid in the sample to obtain analysis results.

[0067] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0068] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0069] Example 1 Screening Primers and Probes

[0070] 1. Screening of primer and probe combinations for ORP (orthopoxvirus), as shown in Table 1:

[0071] Table 1

[0072] SEQ ID NO: Primer probe name sequence 1 ORP-F1 5'-GCCAGAGATATCATAGCCGCTC-3' 2 ORP-F2 5'-AGATATCATAGCCGCTCTTAGAG-3' 3 ORP-F 5'-GATATCATAGCCGCTCTCAG-3' 4 ORP-R1 5'-CAACGACTAACTAATTTGGAAAAAAAGAT-3' 5 ORP-R 5'-CGACTAACTAATTTGGAAAAAAAGAT-3' 6 ORP-P1 5'-ROX-TTTTCCAACCTAAATAGAACGTCATCATTGCGTT-BQ2-3' 7 ORP-P 5'-ROX-TTCCAACCTAAATAGAACGTCATCATTG-BQ2-3'

[0073] By permuting and combining the forward primers (F1 / F2 / F), reverse primers (R1 / R) and probes (P1 / P) of the above ORP (12 groups), the primer and probe groups with high sensitivity and strong specificity were screened. Figure 1 shown.

[0074] Among the 12 groups, the combination of upstream and downstream primers and probes of ORP-F, ORP-P, and ORP-R was the best. Compared with them, the amplification curves of the 8 combinations of ORP-F1, ORP-F2, ORP-R1, ORP-R, ORP-P1, and ORP-P did not show a significant S-shape, indicating that the amplification efficiency of these 8 groups was poor. The fluorescence signal intensity of the primer-probe combination of ORP-F, ORP-P1, and ORP-R1 was low, and the signal of the primer-probe combination of ORP-F, ORP-P1, and ORP-R decreased during amplification of high-concentration samples, and the primer-probe combination of ORP-F, ORP-P, and ORP-R1 was delayed at the last concentration CT. Therefore, the primer-probe combination of ORP-F, ORP-P, and ORP-R was selected as the primer probe for subsequent combined tubes.

[0075] 2. The primer and probe combination screening of IC (internal reference gene) is shown in Table 2:

[0076] Table 2

[0077] SEQ ID NO: Primer probe name sequence 8 IC-F 5'-AGCTGGGGAATGGGACTGAG-3' 9 IC-F2 5'-GGGGAATGGGACTGAG-3' 10 IC-R 5'-GAAAGGAAATTATGGGAAAGCCAG-3' 11 IC-R2 5'-GCTGGGGAATGGGACTGAG-3' 12 IC-P 5'-CY5-CTCCCTGCCGGGGCTGCGTG-BQ2-3'

[0078] By arranging and combining the forward primer (F / F2), reverse primer (R / R2) and probe (P) of the above IC, and performing synchronous amplification with the combination ORP-F, ORP-P, ORP-R selected in 1 above (4 groups), the detection results are as follows Figure 2 As shown, the primer probes of IC-F, IC-R, IC-P and the combination of ORP-F, ORP-P, ORP-R have the highest amplification sensitivity, and the amplification CT value is more than 0.5 earlier than the other three combinations under the low concentration sample. Therefore, IC-F, IC-R, and IC-P are selected as the internal reference gene combination of this reagent.

[0079] 3. Primer and probe combination screening for MPV (monkeypox virus) is shown in Table 3:

[0080] Table 3

[0081] SEQ ID NO: Primer probe name sequence 13 MPV-F 5'-CCACTGATTCAATACGAAAAGACC-3' 14 MPV-F2 5'-TTTAACCGGAATAACATCATCAAAAG-3' 15 MPV-R1 5'-GGAGAAGCGAGAAGTTAATAAAGC-3' 16 MPV-R 5'-CAGTTCCGACGATACTCCTCCT-3' 17 MPV-IP 5'-FAM-ACTATGGTGTACAGTTCCG-MGB-3' 18 MPV-II-P 5'-VIC-CTATGGTTTACAGCTCCA-MGB-3'

[0082] The best MVP amplification sequence combination was screened out by arranging and combining the forward primer (F / F2), reverse primer (R1 / R) and probe (MPV-IP and MPV-II-P) of the above MPV and amplifying together with the combination ORP-F, ORP-P, ORP-R screened in the above 1 and the combination IC-F, IC-R, IC-P screened in the above 2 (4 groups). The detection results are as follows: Figure 3 shown.

[0083] Among them, MPV-F, MPV-R, MPV-IP and MPV-II-P can produce the best curve shape when amplified together with ORP-F, ORP-P1, ORP-R and IC-F, IC-R, IC-P, so MPV-F, MPV-R, MPV-IP were selected for application in subsequent steps.

[0084] After screening the above multiple primer sets, a primer and probe set with high sensitivity and strong specificity was finally screened out, and the mutual interference of multiple primers and probes was avoided, as shown in Table 4. The primer set in Table 4 is used to prepare the kit of the present application.

[0085] Table 4

[0086] SEQ ID NO: Primer probe name sequence 3 ORP-F 5'-GATATCATAGCCGCTCTCAG-3' 5 ORP-R 5'-CGACTAACTAATTTGGAAAAAAAGAT-3' 7 ORP-P 5'-ROX-TTCCAACCTAAATAGAACGTCATCATTG-BQ2-3' 13 MPV-F 5'-CTCATTGATTTTTCGCGGGATA-3' 16 MPV-R 5'-CAGTTCCGACGATACTCCTCCT-3' 17 MPV-IP 5'-FAM-ACTATGGTGTACAGTTCCG-MGB-3' 18 MPV-II-P 5'-VIC-CTATGGTTTACAGCTCCA-MGB-3' 8 IC-F 5'-AGCTGGGGAATGGGACTGAG-3' 10 IC-R 5'-GAAAGGAAATTATGGGAAAGCCAG-3' 12 IC-P 5'-CY5-CTCCCTGCCGGGGCTGCGTG-BQ2-3'

[0087] Example 2 Lyophilized reagent and kit

[0088] The present embodiment provides a kit for detecting orthopoxvirus and monkeypox virus typing, which can be stored at room temperature and includes a lyophilized reagent. The components and working concentrations of the lyophilized reagent are shown in Table 5. The lyophilized reagent contains primers and probes shown in SEQ ID NOs. 3, 5, 7, 13, 16, 17, and 18.

[0089] This embodiment also provides a kit for detecting orthopoxvirus, which can be stored at room temperature and includes a lyophilized reagent. The components and working concentrations of the lyophilized reagent are shown in Table 5. The lyophilized reagent contains primers and probes shown in SEQ ID NOs. 3, 5, and 7.

[0090] This embodiment also provides a kit for detecting monkeypox virus and distinguishing monkeypox virus type I and type II. The kit can be stored at room temperature and includes a lyophilized reagent. The components and working concentrations of the lyophilized reagent are shown in Table 5. The lyophilized reagent contains primers and probes shown in SEQ ID NOs. 13, 16, 17, and 18.

[0091] The preparation of the freeze-dried reagent comprises the following steps:

[0092] 1. Reagent preparation:

[0093] The reaction solution was prepared according to the components and concentrations of each sample in Table 5.

[0094] Table 5

[0095]

[0096] 2. Freeze-drying process

[0097] The reaction solution was divided into eight PCR tubes with a volume of 20 μL and freeze-dried according to the process in Table 6:

[0098] Table 6

[0099]

[0100] The freeze-dried products of samples 1 to 5 are as follows: Figures 4 to 8 The freeze-dried state of sample 1 is full and cake-shaped, without voids or freeze-dried powder, and its freeze-dried effect is better than that of sample 2 and sample 3, and the freeze-dried effects of sample 2 and sample 3 are better than those of sample 4 and sample 5.

[0101] Example 3 Method for detecting orthopoxvirus and monkeypox virus and typing monkeypox virus

[0102] The nucleic acid in the sample was extracted using a commercially available nucleic acid extraction reagent from Shenzhen Pumen Technology Co., Ltd.

[0103] 20 μL of ultrapure water and 5 μL of the nucleic acid sample to be tested were added to the lyophilized reagent prepared in Example 2, and 25 μL of the reaction system was obtained after mixing.

[0104] Real-time fluorescence quantitative PCR amplification was performed using the amplification program in Table 7.

[0105] Table 7

[0106]

[0107] Example 4: Sensitivity of the kit test sample

[0108] The primer-probe combination in Table 4 or the kit for detecting orthopoxvirus and monkeypox virus typing composed of samples 1 to 5 in Example 2 was used to extract sample nucleic acids of monkeypox virus type I, type II, cowpox virus, vaccinia virus, etc. according to Example 3, and after gradient dilution, amplification detection was performed according to Example 3. The minimum detection limit of the kit composed of samples 1 to 3 in Example 2 was lower than that of the kit composed of samples 4 to 5 in Example 2.

[0109] Among them, the results of the detection kit composed of sample 1 in Example 2 are shown in Table 8. All samples of the kit composed of sample 1 in Example 2 can be detected, and the ROX, FAM, and VIC channels can detect 1.50E+02 copies / ml of the virus. Among them, the detection results of cowpox virus and vaccinia virus are shown in Table 8. Fig. 9 As shown, the detection results of monkeypox virus type I are as follows Fig.10 As shown, the detection results of monkeypox virus type II are as follows Fig.11 The results of the detection of monkeypox virus type I and type II are shown in Fig.12 shown.

[0110] Table 8

[0111]

[0112] Example 5 Specificity of the sample detected by this kit

[0113] The primer-probe combination described in Table 4 or the kit composed of samples 1 to 5 in Example 2 was used to detect orthopoxvirus and monkeypox virus typing. Viruses that cause the same or similar clinical symptoms as orthopoxvirus were extracted according to Example 3, and amplification detection was performed according to Example 3. The results showed that the detection results of orthopoxvirus (vaccinia virus, cowpox virus, monkeypox virus, etc.) and monkeypox virus type I and type II were positive, while other non-orthopoxviruses were negative, and all samples could be identified. This shows that the kit composed of samples 1 to 5 in Example 2 has good specificity. Among them, the detection results of the kit composed of sample 1 in Example 2 are shown in Table 9; the detection results of a column of negative samples are shown in Table 9. Fig.13 shown.

[0114] Table 9

[0115]

[0116] Example 6 Stability of the kit stored at 37°C

[0117] The kits for detecting orthopoxvirus and monkeypox virus typing composed of samples 1 to 5 in Example 2 were stored at 37°C and tested in January, March and June. The results showed that the stability of the kit composed of samples 1 to 3 in Example 2 at 37°C was better than that of the kit composed of samples 4 to 5 in Example 2, and the minimum detection limit of the kit after 6 months of storage at 37°C was lower than that of the kit composed of samples 4 to 5 in Example 2. This indicates that the components in samples 1 to 3 in Example 2 of the present application can improve the stability and detection sensitivity of the kit at room temperature.

[0118] Among them, the results of the test kit composed of sample 1 in Example 2 are shown in Tables 10-1 to 10-4: the sensitivity and specificity of the test kit of Example 2 stored at 37°C for 1 to 6 months are almost the same as those at 0 month, indicating that the test kit of the present application has good room temperature stability and can be stored at room temperature and 37°C for at least 6 months.

[0119] Table 10-1

[0120]

[0121]

[0122] Table 10-2

[0123]

[0124]

[0125] Table 10-3

[0126]

[0127]

[0128] Table 10-4

[0129]

[0130]

[0131] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0132] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A primer-probe combination, characterized in that: The primer-probe combination includes at least one of the following (1) to (2): (1) a primer pair for orthopoxvirus having a sequence as shown in SEQ ID NO: 3 and SEQ ID NO: 5, and a probe for orthopoxvirus having a sequence as shown in SEQ ID NO: 7; (2) A primer pair for monkeypox virus with sequences as shown in SEQ ID NO: 13 and SEQ ID NO: 16, a probe for monkeypox virus type I with sequence as shown in SEQ ID NO: 17, and a probe for monkeypox virus type II with sequence as shown in SEQ ID NO:

18.

2. The primer-probe combination according to claim 1, characterized in that: The two ends of the probe are modified with a fluorescent group and a quenching group respectively; Optionally, the fluorescent group is selected from one of FAM, HEX, VIC, CY5, ROX, Texsa Red and Quasar 705, and the quenching group is selected from one of BHQ1, BHQ2, BHQ3, TAMRA and MGB.

3. The primer-probe combination according to claim 2, characterized in that: The probe of the orthopoxvirus and the probe of the monkeypox virus type I and type II are modified with the same or different fluorescent groups and quenching groups; and / or The probes of monkeypox virus type I and type II are modified with different fluorescent groups.

4. The primer-probe combination according to claim 3, characterized in that: The 5' end of the orthopoxvirus probe is modified with a fluorescent group ROX, and the 3' end is modified with a quenching group BQ2; Optionally, the 5' end of the monkeypox virus type I probe is modified with a fluorescent group FAM or VIC, and the 3' end is modified with a quenching group MGB; Optionally, the 5' end of the monkeypox virus type II probe is modified with a fluorescent group FAM or VIC, and the 3' end is modified with a quenching group MGB.

5. The primer-probe combination according to any one of claims 1 to 4, characterized in that: The primer-probe combination also includes a primer pair and a probe of an internal reference gene; Optionally, the sequences of the primer pair of the internal reference gene are shown in SEQ ID NO: 8 and SEQ ID NO: 10, and the sequence of the probe of the internal reference gene is shown in SEQ ID NO: 12; Optionally, the probe of the internal reference gene and the probe of each virus are modified with different fluorescent groups.

6. Use of the primer-probe combination according to any one of claims 1 to 5 in the preparation of a product for detecting orthopoxvirus and / or detecting monkeypox virus and distinguishing between monkeypox virus type I and type II.

7. A virus detection kit, characterized in that: The kit comprises reagents for nucleic acid amplification, and the reagents for nucleic acid amplification comprise the primer-probe combination according to any one of claims 1 to 5.

8. The virus detection kit according to claim 7, characterized in that: The reagents used for nucleic acid amplification also include one or more of Taq DNA polymerase, UNG enzyme, Tris-HCl, MgCl2, KCl and dNTP.

9. The virus detection kit according to claim 7 or 8, characterized in that: The reagent used for nucleic acid amplification is a lyophilized powder reagent, and the reagent used for nucleic acid amplification also includes a lyophilized excipient; Optionally, the lyophilization excipients include one or more of trehalose, mannitol, bovine serum albumin, Tween 80, polyethylene glycol, polyvinyl pyrrolidone, tert-butyl alcohol, lactose and sodium carboxymethyl cellulose.

10. The virus detection kit according to claim 9, characterized in that: The lyophilized powder reagent contains the following components at working concentrations: 0.5U / reaction~2U / reaction Taq DNA polymerase, 0.1U / reaction~0.5U / reaction UNG enzyme, 10mM~80mMTris-HCl, 1.0mM~5.0mM MgCl2, 10mM~100mM KCl, 0.1μM~1μM dNTPs, 2%w / v~5%w / v trehalose, 2%w / v~5%w / v mannitol, 1%w / v~3%w / v bovine serum albumin, 0.1%v / v~0.6%v / v Tween80, 0.5%w / v~2%w / v PEG20000, 0.2%w / v~0.5%w / v PVP40000, 0.1% v / v-2% v / v tert-butyl alcohol, 1% w / v-3% w / v lactose, 1% w / v-3% w / v sodium carboxymethyl cellulose, 50 nM-200 nM of each primer and 50 nM-200 nM of each probe.

11. The virus detection kit according to any one of claims 7 to 8 and 10, characterized in that: The kit also includes one or more of a reconstitution solvent, a negative quality control product, and a positive quality control product.

12. A method for detecting orthopoxviruses for non-diagnostic therapeutic purposes, and / or detecting monkeypox virus and distinguishing between monkeypox virus type I and type II, characterized in that: The method comprises the following steps: Use the primer-probe combination according to any one of claims 1 to 5 or the virus detection kit according to any one of claims 7 to 11 to perform fluorescent quantitative PCR analysis on the nucleic acid in the sample to obtain the analysis result.

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