Primer composition for detecting orthopoxvirus, monkey poxvirus and drug-resistant mutation sites and application

Abstract

The invention provides a primer composition for detecting orthopoxvirus, monkey poxvirus and drug-resistant mutation sites and application, and relates to the technical field of biology, the primer composition comprises a primer probe combination for detecting orthopoxvirus, a primer probe combination for detecting monkey poxvirus and a primer probe combination for detecting drug-resistant mutation sites, the kit can be used for detecting drug-resistant mutation sites H238Q, N267D and A290V of a monkey pox micromolecular antiviral drug Tecovirimat while distinguishing orthopox virus and monkey pox virus. The kit can be applied to real-time fluorescent quantitative PCR detection of the monkey pox virus and the drug-resistant site, is high in sensitivity, strong in specificity and simple and rapid to operate, can also be used for monitoring appearance and transmission of drug-resistant viruses in people, and provides a basis for prevention and control of the viruses.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a primer composition for detecting orthopoxvirus, monkeypox virus and drug-resistant mutation sites and an application thereof. Background Art

[0002] Orthopoxvirus is a large double-stranded DNA virus belonging to the subfamily of Chordatapoxvirus in the Poxviridae family, which can cause widespread zoonotic diseases. Orthopoxviruses include many viruses that can cause human diseases, such as Variola virus, Monkeypox virus (MPV), Cowpox virus and Vaccinia virus. Among them, Monkeypox virus is the most important poxvirus after smallpox virus. Monkeypox is a zoonotic infectious disease caused by infection with monkeypox virus. Monkeypox virus is an enveloped double-stranded DNA virus, belonging to the genus Orthopoxvirus in the Poxviridae family, along with smallpox virus, vaccinia virus and cowpox virus.

[0003] Humans are mainly infected through direct contact with the patient's skin, mucous membranes, blood, body fluids, respiratory droplets, etc., and can also be infected with monkeypox through contact with objects contaminated with monkeypox virus. The population is generally susceptible to monkeypox virus, and there is a certain degree of cross-protection against monkeypox virus after vaccination with smallpox vaccine. Monkeypox is a self-limiting disease, most of which have a good prognosis. Severe cases are common in children and immunocompromised people. Studies have shown that people who have been vaccinated with smallpox vaccine in the past have a certain degree of cross-protection against monkeypox virus. Experience in smallpox treatment shows that smallpox vaccine, cidofovir and brincidofovir, tecovirizumab and vaccinia immune globulin may play a role in the treatment of monkeypox. Tecovirimat (trade name TPOXX, ST-246) is a small molecule broad-spectrum membrane protein inhibitor that targets the VP37 protein of the orthopoxvirus genus (encoded by the F13L gene) and inhibits its activity, blocking its interaction with cellular GTPase and TIP47, thereby preventing the formation of enveloped viruses necessary for intercellular and long-distance transmission of the virus, thereby inhibiting the replication and proliferation of smallpox virus and other orthopoxviruses. Currently, animal models and case reports have confirmed the effectiveness of tecovirimat in monkeypox patients, with good safety. The intercellular and long-distance transmission and virulence of orthopoxviruses are mainly related to the formation of enveloped viruses. Mutations in the gene expressing the VP37 protein of orthopoxviruses will affect the antiviral activity of TPOXX and reduce the effectiveness of the drug. The mutation sites include H238Q, N267D and A290V.

[0004] Virus isolation and identification is the classic method for diagnosing monkeypox virus, which usually takes 3-6 days and has potential biosafety hazards, making it unsuitable for large-scale testing applications; serological diagnosis has low sensitivity and is not easy to differentiate between smallpox, monkeypox and other diseases with rashes; first-generation sequencing is hailed as the gold standard of all generations of sequencing methods, but its detection throughput is low, it takes a long time, and it is prone to contamination; second-generation sequencing has a high detection throughput, but the operation is cumbersome and time-consuming, requiring expensive instruments, well-trained technicians and skilled bioinformatics personnel for computational analysis, and is not suitable for large-scale clinical applications; gene chip technology has made great progress, but there are still problems such as high technical costs, complexity, and low detection sensitivity.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a primer composition for detecting orthopoxvirus, monkeypox virus and drug-resistant mutation sites, so as to solve the technical problem in the prior art that there is a lack of materials that can simultaneously identify orthopoxvirus and monkeypox virus and determine whether the monkeypox virus is resistant to the therapeutic drug tecovirizumab.

[0007] The second object of the present invention is to provide the use of the above-mentioned primer combination in the preparation of a product for detecting or assisting in detecting whether a sample to be tested contains orthopoxvirus and / or monkeypox virus, and / or whether there are drug-resistant mutation sites, or for identifying or assisting in identifying whether the virus to be tested is an orthopoxvirus or monkeypox virus, and / or whether there are drug-resistant mutation sites.

[0008] The third object of the present invention is to provide a reagent.

[0009] A fourth object of the present invention is to provide a test kit.

[0010] A fifth object of the present invention is to provide a method for detecting or assisting in detecting orthopoxvirus and / or monkeypox virus and / or drug-resistant mutation sites for purposes other than disease diagnosis.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0012] In a first aspect, the present invention provides a primer composition for detecting orthopoxvirus, monkeypox virus and drug-resistant mutation sites, including a primer-probe combination for detecting orthopoxvirus, a primer-probe combination for detecting monkeypox virus and a primer-probe combination for detecting drug-resistant mutation sites;

[0013] The primer-probe combination for detecting orthopoxvirus comprises a primer pair 1 and a probe 1, wherein the primer pair 1 is composed of two single-stranded DNAs shown in SEQ ID NO.1 and SEQ ID NO.2, and the probe 1 is a single-stranded DNA shown in SEQ ID NO.3;

[0014] The primer-probe combination for detecting monkeypox virus includes primer pair 2 and probe 2; the primer pair 2 is composed of two single-stranded DNAs shown in SEQ ID NO.4 and SEQ ID NO.5, and the probe 2 is a single-stranded DNA shown in SEQ ID NO.6;

[0015] The primer-probe combination for detecting drug-resistant mutation sites includes primer-probe combination 1 and primer-probe combination 2, and the drug-resistant mutation sites include H238Q, N267D and A290V;

[0016] The primer-probe combination 1 includes at least one of a primer pair 11 and a probe 11 for detecting a drug-resistant mutation site H238Q, a primer pair 12 and a probe 12 for detecting a drug-resistant mutation site N267D, or a primer pair 13 and a probe 13 for detecting a drug-resistant mutation site A290V;

[0017] The primer pair 11 is composed of two single-stranded DNAs shown in SEQ ID NO.7 and SEQ ID NO.8, and the probe 11 is a single-stranded DNA shown in SEQ ID NO.9;

[0018] The primer pair 12 is composed of two single-stranded DNAs shown in SEQ ID NO.10 and SEQ ID NO.11, and the probe 12 is the single-stranded DNA shown in SEQ ID NO.12;

[0019] The primer pair 13 is composed of two single-stranded DNAs shown in SEQ ID NO.13 and SEQ ID NO.14, and the probe 13 is a single-stranded DNA shown in SEQ ID NO.15;

[0020] The primer-probe combination 2 includes at least one of a primer pair 21 and a probe 21 for detecting a drug-resistant mutation site H238Q, a primer pair 22 and a probe 22 for detecting a drug-resistant mutation site N267D, or a primer pair 23 and a probe 23 for detecting a drug-resistant mutation site A290V;

[0021] The primer pair 21 is composed of two single-stranded DNAs shown in SEQ ID NO.16 and SEQ ID NO.17, and the probe 21 is a single-stranded DNA shown in SEQ ID NO.18;

[0022] The primer pair 22 is composed of two single-stranded DNAs shown in SEQ ID NO.19 and SEQ ID NO.20, and the probe 22 is a single-stranded DNA shown in SEQ ID NO.21;

[0023] The primer pair 23 is composed of two single-stranded DNAs shown in SEQ ID NO.22 and SEQ ID NO.23, and the probe 23 is a single-stranded DNA shown in SEQ ID NO.24.

[0024] By comparing the sequences of orthopoxvirus and monkeypox virus published in the GeneBank database, we selected specific viral sequences to design corresponding primer probes, which can detect the drug-resistant mutation sites H238Q, N267D and A290V of the monkeypox small molecule antiviral drug Tecovirimat (TPOXX, ST-246) while distinguishing between orthopoxvirus and monkeypox virus. The sensitivity of the primer combination can reach 5×10 2 copies / mL, with high sensitivity; it can not only distinguish orthopoxvirus and monkeypox virus from each other, but also distinguish them from other viruses of similar species and living in the same environment and other drug-resistant genes, such as measles virus, rubella virus, varicella-zoster virus, syphilis spirochete, human parvovirus B19, etc., proving that the detection method has a high specificity and can accurately distinguish non-detection targets.

[0025] Further, both ends of the probe 1, probe 2, probe 11, probe 12, probe 13, probe 21, probe 22 and probe 23 are respectively labeled with a fluorescent group and a quenching group;

[0026] Preferably, the fluorescent groups labeled with probe 1 and probe 2 are different, the fluorescent groups labeled with probe 11, probe 12 and probe 13 are all different, and the fluorescent groups labeled with probe 21, probe 22 and probe 23 are all different.

[0027] Furthermore, in the primer-probe combination, the molar ratio of the upstream primer, the downstream primer and the probe in the primer pair is 2-8:2-8:1-3.

[0028] In a second aspect, the present invention provides the use of the above-mentioned primer combination in the preparation of a product for detecting or assisting in detecting whether a sample to be tested contains orthopoxvirus and / or monkeypox virus, and / or whether there are drug-resistant mutation sites, or for identifying or assisting in identifying whether the virus to be tested is an orthopoxvirus or monkeypox virus, and / or whether there are drug-resistant mutation sites.

[0029] Further, the sample to be tested includes at least one of a skin lesion surface swab, a pustular fluid or pustular exudate swab, a pustular scab or a pustular scab swab, an oropharyngeal swab, whole blood, plasma, serum, a rectal swab, urine, semen or saliva;

[0030] Preferably, the skin lesion site includes at least one of macules, papules, blisters or pustules.

[0031] In a third aspect, the present invention provides a reagent comprising the above-mentioned primer combination.

[0032] In a fourth aspect, the present invention provides a kit comprising the above-mentioned primer composition or the above-mentioned reagent.

[0033] Furthermore, it also includes at least one of reagents for PCR amplification, positive quality control or negative quality control.

[0034] In a fifth aspect, the present invention provides a method for detecting or assisting in detecting orthopoxvirus and / or monkeypox virus and / or drug-resistant mutation sites, wherein the method is a non-disease diagnosis method. A non-disease diagnosis method is a method that does not directly aim to obtain disease diagnosis results or health conditions of living human or animal bodies.

[0035] The method comprises using the sample DNA to be tested as a template, applying the above primer combination or the above reagent or the above kit to perform multiplex fluorescence quantitative PCR, and determining the result according to the Ct value and amplification curve of each channel:

[0036] If the channel corresponding to the probe 1 meets the positive judgment condition, the channel corresponding to the probe 2 does not meet the positive judgment condition, and the channels corresponding to the probes 11, 12, and 13 do not meet the positive judgment condition, then the sample to be tested contains other orthopoxviruses other than monkeypox virus; if the channels corresponding to the probes 1 and 2 meet the positive judgment condition, and the channels corresponding to the probes 11, 12, and 13 do not meet the positive judgment condition, then the sample to be tested contains monkeypox virus with no mutation in the drug resistance site; if the channels corresponding to the probes 1 and 2 meet the positive judgment condition, and at least one of the channels corresponding to the probes 11, 12, or 13 meets the positive judgment condition, then the sample to be tested contains monkeypox virus with mutation in the drug resistance site; or,

[0037] If the channel corresponding to the probe 1 meets the positive judgment condition, the channel corresponding to the probe 2 does not meet the positive judgment condition, and the ΔCt of the channels corresponding to probes 21, 22 and 23 are all greater than 5, then the sample to be tested contains other orthopoxviruses other than monkeypox virus; if the channels corresponding to the probe 1 and probe 2 meet the positive judgment condition, and the ΔCt of the channels corresponding to probes 21, 22 and 23 are all greater than 5, then the sample to be tested contains monkeypox virus with no mutation in the drug resistance site; if the channels corresponding to the probe 1 and probe 2 meet the positive judgment condition, and the ΔCt of at least one corresponding channel of probe 21, probe 22 or probe 23 is less than or equal to 5, then the sample to be tested contains monkeypox virus with mutation in the drug resistance site.

[0038] The positive judgment condition is: if the sample has S-type amplification in the detection channel and the Ct value is ≤38, it is judged as positive; or, if the sample has S-type amplification in the detection channel and 38<Ct value ≤40, after re-extraction of nucleic acid, there is still S-type amplification and the Ct value is ≤40, it is judged as positive.

[0039] It is applied to the real-time fluorescence quantitative PCR detection of monkeypox virus and drug-resistant sites, and has high sensitivity, strong specificity, simple and fast operation. It can also be used to monitor the emergence and spread of drug-resistant viruses in the population, providing a basis for virus prevention and control. It avoids the cumbersome operations of serology, pathogen culture, first-generation sequencing and other methods.

[0040] Further, if the corresponding channels of the probes 1 and 2 meet the positive judgment conditions, and at least one corresponding channel of the probes 11, 12 or 13 meets the positive judgment conditions, then the monkeypox virus containing the drug-resistant site mutation in the sample to be tested also includes if the corresponding channel of the probe 11 meets the positive judgment conditions, then the drug-resistant mutation site of the monkeypox virus in the sample to be tested is H238Q; if the corresponding channel of the probe 12 meets the positive judgment conditions, then the drug-resistant mutation site of the monkeypox virus in the sample to be tested is N267D; if the corresponding channel of the probe 13 meets the positive judgment conditions, then the drug-resistant mutation site of the monkeypox virus in the sample to be tested is A290V;

[0041] Preferably, if the channels corresponding to the probes 1 and 2 satisfy the positive judgment condition, and the ΔCt of at least one channel corresponding to the probes 21, 22 or 23 is less than or equal to 5, then the monkeypox virus containing the drug-resistant site mutation in the sample to be tested also includes if the ΔCt of the channel corresponding to the probe 21 is less than or equal to 5, then the drug-resistant mutation site of the monkeypox virus in the sample to be tested is H238Q; if the ΔCt of the channel corresponding to the probe 22 is less than or equal to 5, then the drug-resistant mutation site of the monkeypox virus in the sample to be tested is N267D; if the ΔCt of the channel corresponding to the probe 23 is less than or equal to 5, then the drug-resistant mutation site of the monkeypox virus in the sample to be tested is A290V;

[0042] Preferably, the ΔCt=Ct value of the current channel-Ct value of the probe 1 channel.

[0043] The primer composition for detecting orthopoxvirus, monkeypox virus and drug-resistant mutation sites provided by the present invention can detect drug-resistant mutation sites H238Q, N267D and A290V of the monkeypox small molecule antiviral drug Tecovirimat (TPOXX, ST-246) while distinguishing orthopoxvirus from monkeypox virus. It can be applied to real-time fluorescence quantitative PCR detection of monkeypox virus and drug-resistant sites, and has high sensitivity, strong specificity, simple and rapid operation, and can also be used to monitor the emergence and spread of drug-resistant viruses in the population, providing a basis for virus prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 The single-plex PCR screening amplification curve of the primer-probe combination in the different primer compositions provided in Example 3 of the present invention - A tube monkeypox virus and orthopoxvirus - orthopoxvirus;

[0046] Figure 2 The single-plex PCR screening amplification curve of the primer-probe combination in the different primer compositions provided in Example 3 of the present invention - A-tube monkeypox virus and orthopoxvirus-monkeypox virus;

[0047] Figure 3 The primer-probe combination single-plex PCR screening amplification curve of the different primer combinations provided in Example 3 of the present invention - the primer-probe combination of the drug resistance site - tube B - H238Q;

[0048] Figure 4 The primer-probe combination single-plex PCR screening amplification curve of the different primer combinations provided in Example 3 of the present invention - the primer-probe combination of the drug resistance site - tube B - N267D;

[0049] Figure 5 The primer-probe combination single-plex PCR screening amplification curve of the different primer combinations provided in Example 3 of the present invention - the primer-probe combination of the drug resistance site - tube B - A290V;

[0050] Figure 6 The primer-probe combination single-plex PCR screening amplification curve of the different primer combinations provided in Example 3 of the present invention - the primer-probe combination C tube of the drug resistance site - H238Q;

[0051] Figure 7 The primer-probe combination single-plex PCR screening amplification curve of the different primer combinations provided in Example 3 of the present invention - the primer-probe combination D tube of the drug resistance site - N267D;

[0052] Figure 8 The primer-probe combination single-plex PCR screening amplification curve of the different primer combinations provided in Example 3 of the present invention - the primer-probe combination E tube of the drug resistance site - A290V;

[0053] Fig. 9Amplification curve for specific detection of the primer-probe combination in the primer composition provided in Example 4 of the present invention - monkeypox virus and orthopoxvirus detection - tube A;

[0054] Fig.10 A primer-probe combination specific detection amplification curve in the primer composition provided in Example 4 of the present invention - a primer-probe combination for drug resistance sites - tube B;

[0055] Fig.11 Amplification curve of primer-probe combination specific detection in the primer combination provided in Example 4 of the present invention - primer-probe combination C tube, D tube and E tube of drug resistance site;

[0056] Fig.12 The accuracy detection amplification curve of the primer combination provided in Example 5 of the present invention using the monkeypox virus plasmid MP-plasmid as a sample, wherein 1 is the orthopoxvirus of tube A, 2 is the monkeypox virus of tube A, 3 is H38Q of tube C, 4 is N267D of tube D, and 5 is A290V of tube E;

[0057] Fig.13 The accuracy detection amplification curve of the primer combination using H238Q-plasmid as a sample provided in Example 5 of the present invention, wherein 1 is orthopoxvirus in tube A, 2 is monkeypox virus in tube A, 3 is H38Q in tube B, 4 is H38Q in tube C, 5 is N267D in tube D, and 6 is A290V in tube E;

[0058] Fig.14 The accuracy detection amplification curve of the primer combination provided in Example 5 of the present invention using N267D-plasmid as a sample, wherein 1 is tube A orthopoxvirus, 2 is tube A monkeypox virus, 3 is tube B N267D, 4 is tube C H38Q, 5 is tube D N267D, and 6 is tube E A290V;

[0059] Fig.15 The accuracy detection amplification curve of the primer combination using A290V-plasmid as a sample provided in Example 5 of the present invention, wherein 1 is orthopoxvirus in tube A, 2 is monkeypox virus in tube A, 3 is A290V in tube B, 4 is H38Q in tube C, 5 is N267D in tube D, and 6 is A290V in tube E;

[0060] Fig.16 The accuracy detection amplification curve of the primer combination using the vaccinia virus plasmid as a sample provided in Example 5 of the present invention, wherein 1 is the A-tube orthopoxvirus;

[0061] Fig.17 The accuracy detection amplification curve of the primer combination provided in Example 5 of the present invention using the smallpox virus plasmid as a sample, wherein 1 is the A-tube orthopoxvirus;

[0062] Fig.18The accuracy detection amplification curve of the primer combination using vaccinia virus plasmid as a sample provided in Example 5 of the present invention, wherein 1 is A-tube orthopoxvirus;

[0063] Fig.19 Amplification curve for sensitivity detection of samples with different concentration gradients - monkeypox virus and orthopoxvirus detection - tube A provided in Example 6 of the present invention;

[0064] Fig. 20 The sensitivity detection amplification curve of samples with different concentration gradients provided in Example 6 of the present invention - the primer-probe combination of drug resistance sites - tube B;

[0065] Fig.21 The C tube for the sensitivity detection amplification curve of samples with different concentration gradients - the primer-probe combination of drug resistance sites provided in Example 6 of the present invention;

[0066] Fig. 22 The tube D for the sensitivity detection amplification curve of samples with different concentration gradients - the primer-probe combination for drug resistance sites provided in Example 6 of the present invention;

[0067] Fig.23 The sensitivity detection amplification curve of samples with different concentration gradients provided in Example 6 of the present invention - the primer probe combination E tube of drug resistance site, wherein 1 to 3 are 10 6 , 5×10 4 , 5×10 3 Concentration, 4 to 6 are all 5×10 2 Concentration, in Copies / mL. DETAILED DESCRIPTION

[0068] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0069] Example 1 Design and screening of specific primer combinations

[0070] 1. Design of primer and probe combinations for detecting monkeypox virus

[0071] By comparing the orthopoxvirus sequences published in the GeneBank database, the F3L gene was determined as the target for monkeypox virus detection. Taking the whole genome of monkeypox virus NC_003310.1 as an example, the F3L gene is located at base positions 48048-48509. Considering that the genes of members of the poxvirus genus have certain homology, the present invention analyzed and compared the screened region with smallpox virus, cowpox virus, etc., and designed a primer and probe combination for monkeypox virus detection based on site differences.

[0072] 2. Design of primer and probe combinations for detection of drug-resistant mutation sites H238Q, N267D and A290V of orthopoxvirus and tecovirizumab

[0073] The present invention compares the orthopoxvirus sequences published on the GeneBank database, selects the expression gene F13L of the VP37 protein as a target to design the detection targets of the drug-resistant mutation sites H238Q, N267D, and A290V of orthopoxvirus and tecoviridae. The orthopoxvirus takes the monkeypox virus full genome NC_003310.1 as an example, and its F13L gene is located at the base positions of 48048-48509. Other orthopoxviruses are cowpox virus NC_003663.2, smallpox virus NC_001611.1, vaccinia virus NC_006998.1, etc., and the drug-resistant mutation sites H238Q, N267D, and A290V take the monkeypox virus F13L gene sequences OP890563, OP680505, and OP748968 as examples. The above-listed genes and other orthopoxvirus sequences found in Genbank were imported into Mega7 software, and the conservative sequence segments that can be used for primer probe design were obtained by comparison analysis, and then respectively imported into Oligo 7 software to design primer probes. In the design process, the problem of co-amplification of primer probes of different target genes in one reaction system was fully considered. Therefore, when designing primer probes, the consistency of Tm value and the homogenization of GC content should be considered, and the occurrence of hairpin structures and primer dimers should be avoided as much as possible to ensure the probability of simultaneous amplification of different primer probes in the later stage.

[0074] According to the site differences, two sets of primer-probe combinations were designed, based on the MGB probe method and the ARMS-PCR method, and a template plasmid was constructed at the same time.

[0075] 3. Perform BLAST analysis on the amplification primer probe;

[0076] All primer and probe sequences were subjected to BLAST analysis in Genbank, and primer and probe with good specificity were determined as the primer and probe scheme for experimental verification.

[0077] 4. Perform single-plex PCR validation on the primer probes used for each target detection to determine the usability of the primer probes, including specificity and sensitivity assessments.

[0078] 5. Since multiple sets of primer probe combinations are required, there is a high possibility that different primer probes will be cross-linked and unable to be co-amplified. For primer probes that do not work, new primer probes need to be designed repeatedly according to steps 2-4 to replace them. Finally, the specific primer probe sequences provided by the present invention are obtained, as shown in Table 1 and Table 2.

[0079] Table 1 First primer composition

[0080]

[0081] Table 2 Second primer composition

[0082]

[0083]

[0084] Example 2 Preparation of Multiplex Fluorescence Quantitative PCR Detection Kit

[0085] 1) Preparation of nucleic acid amplification reaction solution

[0086] The nucleic acid amplification reaction solution contains the corresponding concentration of PCR buffer, Mg 2+ , dNTPs, primer combination, RNase-free water. Wherein, the primer combination is any primer-probe combination in tube A, tube B, tube C, tube D or tube E. Wherein, tube A includes primer pair 1 and probe 1 and primer pair 2 and probe 2; tube B includes primer pair 11 and probe 11, primer pair 12 and probe 12 and primer pair 13 and probe 13; tube C includes primer pair 21 and probe 21; tube D includes primer pair 22 and probe 22; tube E includes primer pair 23 and probe 23. The concentration of each primer in each tube of primer-probe mixture can be 200-800nM, and the concentration of each probe is 100-300nM. In the embodiment of the present invention, the concentration of each primer is 400nM, and the concentration of each probe is 150nM.

[0087] Tube system A is used to detect orthopoxvirus and monkeypox virus, tube system B is based on the MGB probe method to detect mutation sites H238Q, N267D and A290V, tube system C is based on the ARMS-PCR method to detect mutation site H238Q, tube system D is based on the ARMS-PCR method to detect mutation site N267D, and tube system E is based on the ARMS-PCR method to detect mutation site A290V. Specifically, tube system A and tube system B can be used to detect orthopoxvirus, monkeypox virus and drug-resistant mutation sites, or tube system A can be used to detect orthopoxvirus, monkeypox virus and drug-resistant mutation sites with tube systems C to E.

[0088] Prepared according to the following formula (final concentration): 30 mM Tris-HCl (pH = 8.3), 100 mM KCl, 1.5 mM dNTPs, 4 mM MgCl 2 , each primer 0.4μM, each probe 0.15μM.

[0089] 2) Preparation of positive control

[0090] Dilute the positive plasmid with TE to a final concentration of 10 7 copies / mL of solution.

[0091] The monkeypox virus plasmid MP-plasmid sequence (SEQ ID NO.49) contains the full-length F3L gene (NC_003310.1:48048-48509, 462bp) and the full-length F13L gene (NC_003310.1:41104-42222, 1119bp), which is used for monkeypox virus and orthopoxvirus detection.

[0092] The vaccinia virus plasmid CP-plasmid sequence (SEQ ID NO.50) contains the full length of the F3L gene (NC_003663.2: 64465-64926, 462bp) and the full length of the F13L gene (NC_003663.2: 57512-58630, 1119bp), which is used for orthopoxvirus detection, and the drug-resistant mutation sites H238Q, N267D, and A290V of monkeypox virus and tecoviridad should be negative.

[0093] The smallpox virus plasmid VariolaP-plasmid sequence (SEQ ID NO.51) contains the full length of the F3L gene (NC_001611.1: 38632-39093, 462bp) and the full length of the F13L gene (NC_001611.1: 31694-32812, 1119bp), which is used for orthopoxvirus detection, and the drug-resistant mutation sites H238Q, N267D, and A290V of monkeypox virus and tecoviridad should be negative.

[0094] The vaccinia virus plasmid VacciniaP-plasmid sequence (SEQ ID NO.52) contains the full length of the F3L gene (NC_006998.1:47780-48241, 462bp) and the full length of the F13L gene (NC_006998.1:40831-41949, 1119bp), which is used for orthopoxvirus detection, and the drug-resistant mutation sites H238Q, N267D, and A290V of monkeypox virus and tecoviridad should be negative.

[0095] The drug-resistant mutation site plasmid H238Q-plasmid sequence (SEQ ID NO.53), N267D-plasmid sequence (SEQ ID NO.54) and A290V-plasmid sequence (SEQ ID NO.55) of the monkeypox virus Tecoviridae all contain the full length of the monkeypox F3L gene (NC_003310.1:48048-48509, 462bp) and the full length of the F3L gene, which are derived from GeneBank OP890564.1, OP890572.1, and OP748968.1, respectively, corresponding to the above drug-resistant mutation sites, and are used for drug-resistant mutation site detection.

[0096] 3) Preparation of negative control: TE solution was used as negative control.

[0097] 4) Mixed enzyme solution

[0098] The reverse transcriptase (3U / μL), DNA polymerase (2U / μL), and UNG enzyme (0.5U / μL) are in the form of a mixed solution of enzymes in the kit. Generally speaking, the DNA polymerase used is a thermostable Taq DNA polymerase.

[0099] 5) The composition of the kit is shown in Table 3.

[0100] Table 3 Kit composition

[0101] Composition (50tests / box) volume Nucleic acid amplification reaction solution 900μL×1 Mixed enzyme solution 100μL Positive Control 100μL×1 Negative Control 1000μL×1

[0102] Method for using the kit of this example

[0103] 1) Sample extraction

[0104] The magnetic bead method was used to extract viral nucleic acids from clinical samples such as surface swabs of skin (mucosal) lesions such as macules, papules, blisters or pustules, acne fluid or acne exudate swabs, acne scabs or acne scab swabs, oropharyngeal swabs, whole blood, plasma, serum, rectal swabs, urine, semen, saliva, etc. according to the corresponding requirements and steps in the viral DNA extraction kit.

[0105] 2) Preparation of reaction system (25 μL system)

[0106] System preparation: Take out the reagents from the kit and thaw at room temperature. When the reagents are completely thawed, mix them by inversion and centrifuge them instantly. If the number of samples to be tested is n (n = number of samples + positive control + negative control), prepare the system according to n+1 reactions. The reaction system preparation is shown in Table 4.

[0107] Table 4

[0108] Reagents Amount of 1 reaction system The amount of n+1 reaction systems Nucleic acid amplification reaction solution 18μL 18μL×(n+1) Enzyme mixture 2μL 2μL×(n+1)

[0109] System packaging: After mixing and centrifuging the above reaction solution, dispense 20 μL per tube into PCR tubes suitable for fluorescent PCR instrument.

[0110] 3) Add sample

[0111] Take 5 μL of DNA sample extracted in step 1) and add it to the aliquoted PCR tubes. Add 5 μL of negative control to the negative control reaction tube and 5 μL of corresponding template to the positive control reaction tube, fasten the tube cap, flick to mix, centrifuge briefly to mix, and move to the amplification area.

[0112] 4) Detection

[0113] The detection was performed on a real-time fluorescence PCR instrument, and the eight PCR tubes were placed in the fluorescence quantitative PCR instrument, and the names of each well were set. The amplification conditions are shown in Table 5.

[0114] Table 5

[0115]

[0116] 5) Result analysis

[0117] Read the test results directly according to the type of fluorescein in the amplification curve. The baseline and threshold setting principles are adjusted according to the instrument noise situation, and the threshold is set based on the instrument's automatic value or just above the highest point of the negative control amplification curve.

[0118] Quality control standard: If the negative control has no amplification curve and the positive control has an S-shaped amplification curve in both detection channels, the experiment is established. Otherwise, the experimental results are considered invalid.

[0119] 6) Result description and judgment

[0120] If the sample has S-type amplification in the detection channel and the Ct value is ≤38, it is determined according to Table 6 or Table 7 based on the fluorescence channel corresponding to the detection target; b) If the sample has S-type amplification in the detection channel and 38<Ct value≤40, it is determined to be an uncertain sample and the nucleic acid needs to be re-extracted for testing; If the re-tested sample still has S-type amplification in the detection channel and the Ct value is ≤40, it is determined according to the following table based on the fluorescence channel corresponding to the detection target, otherwise it is determined to be negative; c) If the sample has no obvious S-type amplification curve in the detection channel, but a Ct value is reported, it is still determined to be negative.

[0121] Table 6 Determination table of mutation sites detected based on MGB probe method

[0122]

[0123] Table 7 Determination table of mutation sites detected based on ARMS-PCR method

[0124]

[0125] For the same test sample, △Ct = the target Ct value of the current channel detection - the Ct value of the gene of the FAM channel (orthopoxvirus) of tube A. "+" indicates a positive result, and "-" indicates a negative result. If the sample has S-type amplification in the detection channel and the Ct value is ≤38, it is judged as a positive result; or, if the sample has S-type amplification in the detection channel, and 38 < Ct value ≤40, and there is still S-type amplification after re-extraction of nucleic acid, and the Ct value is ≤40, it is judged as a positive result, otherwise it is a negative result.

[0126] Example 3 Single-plex PCR screening of the primer combination of the present invention

[0127] In order to screen the optimal primer-probe combination that can be used in the kit of the present invention, monkeypox virus plasmid MP-plasmid, drug-resistant mutation site plasmids H238Q-plasmid, N267D-plasmid, A290V-plasmid were detected respectively, and one negative sample was used to screen the primer-probe combination. The results are shown in Table 8 and Figures 1 to 8 As shown, the primer-probe combination in the first primer combination and the second primer combination detected no amplification in the negative sample, had good specificity, detected the corresponding plasmid, the primer-probe group in the first primer combination had a Ct value about 3-4 earlier than the primer-probe group in the second primer combination, and had a more obvious S-shaped amplification curve. Therefore, the first primer combination was selected for the kit provided in Example 2 of the present invention.

[0128] Table 8

[0129]

[0130] Example 4 Specificity Verification

[0131] This example verifies the specificity of each primer probe group in the first primer combination, extracts nucleic acid from three human negative samples, measles virus, rubella virus, herpes simplex virus, varicella-zoster virus, syphilis spirochetes and human parvovirus B19 (all inactivated samples), and detects them using the detection reaction system provided in Example 2. The results are shown in Tables 9 and Figures 9 to 11 The results showed that only the positive control was amplified, while the others were not amplified, indicating that the primer combination of the present invention has good specificity.

[0132] Table 9 Specificity test results

[0133]

[0134]

[0135] Example 5 Accuracy Verification

[0136] This example verifies the accuracy of each primer probe group in the first primer combination, and detects monkeypox virus plasmid MP-plasmid, cowpox virus plasmid CP-plasmid, smallpox virus plasmid VariolaP-plasmid, vaccinia virus plasmid VacciniaP-plasmid, drug resistance mutation site plasmid H238Q-plasmid, N267D-plasmid, A290V-plasmid, and the above plasmids are measured for concentration, and then converted into Copies / mL units after calculation, and diluted to 10 5 Copies / mL were used for detection. The results are shown in Table 10 and Figures 12 to 18 As shown, it can be seen that the test results show that monkeypox virus and orthopoxvirus are positive, and the results of drug resistance site mutation are accurate. The results show that this kit can accurately detect monkeypox virus and can detect whether the drug resistance site of the virus Tecovir is mutated.

[0137] Table 10 Accuracy test results

[0138]

[0139] Example 6: Sensitivity verification of the kit of the present invention

[0140] This example verifies the sensitivity of each primer probe group in the first primer combination, and measures the concentration of monkeypox virus plasmid MP-plasmid, drug-resistant mutation site plasmid H238Q-plasmid, N267D-plasmid, and A290V-plasmid, and then converts it into Copies / mL unit after calculation, and dilutes the relevant positive plasmid gradient. The plasmid gradient was detected using the kit of the present invention, and the results are shown in Table 11 and Figure 19 to Figure 23 As shown, the results show that this kit has high sensitivity.

[0141] Table 11 Detection limit verification results

[0142]

[0143] Example 7 Shelf Life Test

[0144] Take 10 5 Copies / mL of the mixed template was used as the test sample for evaluation. On day 0, it was divided into 9 portions and frozen in a -70°C refrigerator. The assembled test kit was stored at -20°C, and the test kits of 0, 10, 15, 30, 60, 90, 120, 150, 180 and 360 days were taken for shelf life test. The shelf life test results are shown in Table 12.

[0145] Table 12 Shelf life test results

[0146] Shelf life Check the effectiveness of each goal Day 0 All effective Day 10 All effective Day 15 All effective Day 30 All effective Day 60 All effective Day 90 All effective Day 120 All effective Day 150 All effective Day 180 All effective Day 360 All effective

[0147] As shown in Table 12, the test kit was stored in a -20°C refrigerator, and all targets were effective in detection at different storage periods. The experimental results showed that the shelf life of the test kit can reach 1 year.

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer composition for detecting orthopoxvirus, monkeypox virus and drug-resistant mutation sites, characterized in that: It includes a primer-probe combination for detecting orthopoxvirus, a primer-probe combination for detecting monkeypox virus, and a primer-probe combination for detecting drug-resistant mutation sites; The primer-probe combination for detecting orthopoxvirus comprises a primer pair 1 and a probe 1, wherein the primer pair 1 is composed of two single-stranded DNAs shown in SEQ ID NO.1 and SEQ ID NO.2, and the probe 1 is a single-stranded DNA shown in SEQ ID NO.3; The primer-probe combination for detecting monkeypox virus includes primer pair 2 and probe 2; the primer pair 2 is composed of two single-stranded DNAs shown in SEQ ID NO.4 and SEQ ID NO.5, and the probe 2 is a single-stranded DNA shown in SEQ ID NO.6; The primer-probe combination for detecting drug-resistant mutation sites includes primer-probe combination 1 and primer-probe combination 2, and the drug-resistant mutation sites include H238Q, N267D and A290V; The primer-probe combination 1 includes at least one of a primer pair 11 and a probe 11 for detecting a drug-resistant mutation site H238Q, a primer pair 12 and a probe 12 for detecting a drug-resistant mutation site N267D, or a primer pair 13 and a probe 13 for detecting a drug-resistant mutation site A290V; The primer pair 11 is composed of two single-stranded DNAs shown in SEQ ID NO.7 and SEQ ID NO.8, and the probe 11 is a single-stranded DNA shown in SEQ ID NO.9; The primer pair 12 is composed of two single-stranded DNAs shown in SEQ ID NO.10 and SEQ ID NO.11, and the probe 12 is the single-stranded DNA shown in SEQ ID NO.12; The primer pair 13 is composed of two single-stranded DNAs shown in SEQ ID NO.13 and SEQ ID NO.14, and the probe 13 is a single-stranded DNA shown in SEQ ID NO.15; The primer-probe combination 2 includes at least one of a primer pair 21 and a probe 21 for detecting a drug-resistant mutation site H238Q, a primer pair 22 and a probe 22 for detecting a drug-resistant mutation site N267D, or a primer pair 23 and a probe 23 for detecting a drug-resistant mutation site A290V; The primer pair 21 is composed of two single-stranded DNAs shown in SEQ ID NO.16 and SEQ ID NO.17, and the probe 21 is a single-stranded DNA shown in SEQ ID NO.18; The primer pair 22 is composed of two single-stranded DNAs shown in SEQ ID NO.19 and SEQ ID NO.20, and the probe 22 is a single-stranded DNA shown in SEQ ID NO.21; The primer pair 23 is composed of two single-stranded DNAs shown in SEQ ID NO.22 and SEQ ID NO.23, and the probe 23 is a single-stranded DNA shown in SEQ ID NO.

24.

2. The primer composition according to claim 1, characterized in that Both ends of the probe 1, probe 2, probe 11, probe 12, probe 13, probe 21, probe 22 and probe 23 are respectively labeled with a fluorescent group and a quenching group; Preferably, the fluorescent groups labeled with probe 1 and probe 2 are different, the fluorescent groups labeled with probe 11, probe 12 and probe 13 are all different, and the fluorescent groups labeled with probe 21, probe 22 and probe 23 are all different.

3. The primer composition according to claim 1 or 2, characterized in that: In the primer-probe combination, the molar ratio of the upstream primer, the downstream primer and the probe in the primer pair is 2-8:2-8:1-3.

4. Use of the primer combination described in any one of claims 1 to 3 in the preparation of a product for detecting or assisting in detecting whether a sample to be tested contains orthopoxvirus and / or monkeypox virus, and / or whether there are drug-resistant mutation sites, or for identifying or assisting in identifying whether the virus to be tested is an orthopoxvirus or monkeypox virus, and / or whether there are drug-resistant mutation sites.

5. The use according to claim 4, characterized in that: The sample to be tested includes at least one of a skin lesion surface swab, a pustular fluid or pustular exudate swab, a pustular scab or a pustular scab swab, an oropharyngeal swab, whole blood, plasma, serum, a rectal swab, urine, semen or saliva; Preferably, the skin lesion site includes at least one of macules, papules, blisters or pustules.

6. A reagent, characterized in that The invention comprises the primer composition according to any one of claims 1 to 3.

7. A kit, characterized in that: The method comprises the primer composition according to any one of claims 1 to 3, or the reagent according to claim 6.

8. The kit according to claim 7, characterized in that Also included are at least one of reagents for PCR amplification, positive quality control, or negative quality control.

9. A method for detecting or assisting in detecting orthopoxvirus and / or monkeypox virus and / or drug-resistant mutation sites for purposes other than disease diagnosis, characterized in that: The method comprises using the sample DNA to be tested as a template, applying the primer combination according to any one of claims 1 to 3, the reagent according to claim 6, or the kit according to claim 7 or 8 to perform multiplex fluorescence quantitative PCR, and determining the result according to the Ct value and amplification curve of each channel: If the channel corresponding to the probe 1 meets the positive judgment condition, the channel corresponding to the probe 2 does not meet the positive judgment condition, and the channels corresponding to the probes 11, 12, and 13 do not meet the positive judgment condition, then the sample to be tested contains other orthopoxviruses other than monkeypox virus; if the channels corresponding to the probes 1 and 2 meet the positive judgment condition, and the channels corresponding to the probes 11, 12, and 13 do not meet the positive judgment condition, then the sample to be tested contains monkeypox virus with no mutation in the drug resistance site; if the channels corresponding to the probes 1 and 2 meet the positive judgment condition, and at least one of the channels corresponding to the probes 11, 12, or 13 meets the positive judgment condition, then the sample to be tested contains monkeypox virus with mutation in the drug resistance site; or, If the channel corresponding to the probe 1 meets the positive judgment condition, the channel corresponding to the probe 2 does not meet the positive judgment condition, and the ΔCt of the channels corresponding to the probes 21, 22, and 23 are all greater than 5, then the sample to be tested contains other orthopoxviruses other than monkeypox virus; if the channels corresponding to the probes 1 and 2 meet the positive judgment condition, and the ΔCt of the channels corresponding to the probes 21, 22, and 23 are all greater than 5, then the sample to be tested contains monkeypox virus with no mutation in the drug resistance site; if the channels corresponding to the probes 1 and 2 meet the positive judgment condition, and the ΔCt of at least one corresponding channel of the probes 21, 22, or 23 is less than or equal to 5, then the sample to be tested contains monkeypox virus with mutation in the drug resistance site; The positive judgment condition is: if the sample has S-type amplification in the detection channel and the Ct value is ≤38, it is judged as positive; or, if the sample has S-type amplification in the detection channel and 38<Ct value ≤40, after re-extraction of nucleic acid, there is still S-type amplification and the Ct value is ≤40, it is judged as positive.

10. The method according to claim 9, characterized in that If the channels corresponding to the probes 1 and 2 satisfy the positive determination conditions, and at least one channel corresponding to the probes 11, 12 or 13 satisfies the positive determination conditions, then the monkeypox virus containing the drug-resistant site mutation in the sample to be tested also includes: if the channel corresponding to the probe 11 satisfies the positive determination conditions, the drug-resistant mutation site of the monkeypox virus in the sample to be tested is H238Q; if the channel corresponding to the probe 12 satisfies the positive determination conditions, the drug-resistant mutation site of the monkeypox virus in the sample to be tested is N267D; if the channel corresponding to the probe 13 satisfies the positive determination conditions, the drug-resistant mutation site of the monkeypox virus in the sample to be tested is A290V; Preferably, if the channels corresponding to the probes 1 and 2 satisfy the positive judgment condition, and the ΔCt of at least one channel corresponding to the probes 21, 22 or 23 is less than or equal to 5, then the monkeypox virus containing the drug-resistant site mutation in the sample to be tested also includes if the ΔCt of the channel corresponding to the probe 21 is less than or equal to 5, then the drug-resistant mutation site of the monkeypox virus in the sample to be tested is H238Q; if the ΔCt of the channel corresponding to the probe 22 is less than or equal to 5, then the drug-resistant mutation site of the monkeypox virus in the sample to be tested is N267D; if the ΔCt of the channel corresponding to the probe 23 is less than or equal to 5, then the drug-resistant mutation site of the monkeypox virus in the sample to be tested is A290V; Preferably, the ΔCt=Ct value of the current channel-Ct value of the probe 1 channel.

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