A primer and fluorescent probe for monkeypox virus detection

Abstract

The application discloses primers and fluorescent probes for detection of monkeypox virus. The nucleic acid sequence of the primers comprises sequences shown in SEQ ID NO. 1-SEQ ID NO. 4; and the nucleic acid sequence of the fluorescent probes comprises sequences shown in SEQ ID NO. 5-SEQ ID NO. 6. Two pairs of primers and corresponding fluorescent probes are designed creatively, which can specifically detect a new target gene B21R in the monkeypox virus DNA, and the target site has conservation and specificity. In addition, a double fluorescent PCR method is established, which can further improve the specificity and accuracy of the monkeypox virus detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology and relates to a primer and a fluorescent probe for detecting monkeypox virus. BACKGROUND

[0002] Monkeypox is an important disease with global public health. According to the WHO's recommendation, the detection of monkeypox virus should adopt the polymerase chain reaction (PCR) method, and the immunization method for detecting antigen is not recommended because the antigen of monkeypox virus and other related orthopoxviruses have immunological cross. Therefore, the detection method based on fluorescent PCR has both accuracy and sensitivity, and is the preferred method for laboratory detection.

[0003] At present, the latest "Monkeypox Prevention and Control Technical Guidelines" published in China and the method of the United States CDC both use fluorescent PCR to detect the single gene target of monkeypox virus. The target gene detected in the guidelines of China is F3L gene, but it can be found from sequence comparison that F3L gene does not have specificity, the primer and probe sequence is basically the same as the allele of cowpox virus, and is highly similar to the DNA of rabbitpox, camelpox, equinepox and other animal orthopoxviruses, which may produce false positive results. The target gene detected by the United States CDC is consistent with the target gene reported in the previous literature (Li et al. J Clin Virol. 2006 Jul; 36(3); Li et al. Virol Methods. 2010 Oct; 169(1)), and according to the literature research (Li et al. Virol Methods. 2010 Oct; 169(1)), the specificity of this target gene fragment is good, and there is no positive cross with other orthopoxvirus DNA. However, due to the long DNA sequence of monkeypox virus (about 198k), the area covered by the single gene target is too small, and if the virus has subsequent mutations, false negative results may be produced due to off-target effect, and once the single gene target fails, there is no other target point for verification and retesting.

[0004] CN115161414A discloses a monkeypox virus specific detection target and its oligonucleotide and kit, including specific primers and probes designed for the F3L gene region in the genome of monkeypox virus. After rapidly extracting nucleic acid in the sample, the monkeypox virus nucleic acid is detected by fluorescent PCR technology. This method is simple to operate, and can specifically distinguish the cowpox virus with high sequence similarity by using TaqMan probe to detect the target gene of monkeypox virus, so as to avoid false detection and missed detection.

[0005] CN114752711A discloses a composition, kit, method for detecting monkeypox virus and typing and purposes thereof, using the composition of the application, the monkeypox virus can be detected rapidly with a sensitivity of 200 copies / mL, and the detection and typing of the monkeypox virus can be realized in the reaction system, so that different monkeypox branches can be distinguished, and treatment and prevention can be more targeted.

[0006] In summary, in order to solve the problems of the target gene F3L of monkeypox virus detection not having specificity, the region covered by single gene target being small, being easy to produce off-target reaction, and being difficult to retest, it is necessary to develop a primer and fluorescent probe for monkeypox virus detection. SUMMARY

[0007] In view of the deficiencies of the prior art and actual needs, the present application provides a primer and fluorescent probe for monkeypox virus detection, which solves the problems of small region covered by single gene target, easy to produce off-target reaction, and difficult to retest.

[0008] To achieve the purpose of the application, the following technical solutions are adopted:

[0009] In a first aspect, the present application provides a primer and fluorescent probe for monkeypox virus detection, the nucleic acid sequence of the primer comprises the sequence shown in SEQ ID NO. 1-SEQ ID NO. 4; the nucleic acid sequence of the fluorescent probe comprises the sequence shown in SEQ ID NO. 5-SEQ ID NO. 6.

[0010] The present application creatively designs two pairs of primers and corresponding fluorescent probes, which can specifically detect the new target gene B21R in monkeypox virus DNA, and the target site has conservation and specificity; in addition, a double fluorescent PCR method is established, which can further improve the specificity and accuracy of monkeypox virus detection.

[0011] SEQ ID NO. 1: CACTTCCTCCTAAACCAGTCCA.

[0012] SEQ ID NO. 2: TGGTTGATCTATAGGAGGAGGT.

[0013] SEQ ID NO. 3: GTTAATACTGGCAGTTCTATTGTGTC.

[0014] SEQ ID NO. 4: TGTTGAGGAAGTGGATTACGGA.

[0015] SEQ ID NO. 5: AACTTCCCCCTAGACCAGTAGGT.

[0016] SEQ ID NO. 6: AGACGACGACCTAGACGCCCT.

[0017] Preferably, the 5' end of the fluorescent probe contains a fluorescent group, and the 3' end contains a quenching group.

[0018] Preferably, the fluorescent group includes any one or a combination of at least two of VIC, FAM, or CY5.

[0019] Preferably, the quenching group includes BHQ1 and / or BHQ2.

[0020] In a second aspect, the present application provides use of the primer and fluorescent probe for monkeypox virus detection in the first aspect in the preparation of a product for monkeypox virus detection.

[0021] In a third aspect, the present application provides a kit for monkeypox virus detection, which comprises the primer and probe for monkeypox virus detection in the first aspect.

[0022] In a fourth aspect, the present application provides use of the primer and probe for monkeypox virus detection in the first aspect in the detection of monkeypox virus.

[0023] In a fifth aspect, the present application provides a method for monkeypox virus detection for non-disease diagnosis and / or treatment purposes, which comprises:

[0024] Using the nucleic acid of the sample to be tested as a template, performing fluorescent PCR amplification using the primer and fluorescent probe for monkeypox virus detection in the first aspect, and judging according to the fluorescent PCR amplification result.

[0025] Preferably, the sample to be tested includes any one or a combination of at least two of pustule fluid, scab, swab of lesion, throat swab, whole blood, or serum.

[0026] Preferably, the amplification target of the primer includes a monkeypox virus gene and a reference gene.

[0027] Preferably, the monkeypox virus gene includes B21R and J2R, and the reference gene includes a human RNaseP gene.

[0028] Preferably, the amplification procedure of the fluorescent PCR amplification is as follows:

[0029] (1) 93-95℃, 3-10min; (2) 93-95℃, 10-50s; (3) 50-70℃, 0.5-3min; 20-50 cycles.

[0030] The specific point value in the above 93-95 can be selected as 93, 94, 95, etc.

[0031] The specific point value in the above 3-10 can be selected from 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0032] The specific point value in the above 10-50 can be selected from 10, 15, 20, 30, 40, 45, 48, 50, etc.

[0033] The specific point value in the above 50-70 can be selected from 50, 55, 60, 65, 68, 70, etc.

[0034] The specific point value in the above 0.5-3 can be selected from 0.5, 1, 1.5, 2, 2.5, 3, etc.

[0035] The specific point value in the above 20-50 can be selected from 20, 25, 30, 35, 40, 45, 48, 50, etc.

[0036] Compared with the prior art, the present application has the following beneficial effects:

[0037] The present application creatively designs two pairs of primers and corresponding fluorescent probes, which can specifically detect the new target gene B21R in monkeypox virus DNA, and the target site has conservation and specificity; in addition, a double fluorescent PCR method is established, which can further improve the specificity and accuracy of monkeypox virus detection. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1A Comparison chart of monkeypox virus B21R gene DNA (1-280 bases) and other similar orthopoxvirus homologous gene sequences;

[0039] Figure 1B Comparison chart of monkeypox virus B21R gene DNA (281-494 bases) and other similar orthopoxvirus homologous gene sequences;

[0040] Figure 1C Comparison chart of monkeypox virus B21R gene DNA (495-715 bases) and other similar orthopoxvirus homologous gene sequences;

[0041] Figure 1D Comparison chart of monkeypox virus B21R gene DNA (716-977 bases) and other similar orthopoxvirus homologous gene sequences;

[0042] Figure 1E Comparison chart of monkeypox virus B21R gene DNA (978-1198 bases) and other similar orthopoxvirus homologous gene sequences;

[0043] Figure 1F Comparison chart of monkeypox virus B21R gene DNA (1199-1435 bases) and other similar orthopoxvirus homologous gene sequences;

[0044] Figure 1G Alignment of monkeypox virus B21R gene DNA (bases 1436-1721) and other orthopoxvirus homologous gene sequences;

[0045] Figure 1H Alignment of monkeypox virus B21R gene DNA (bases 1722-1966) and other orthopoxvirus homologous gene sequences;

[0046] Figure 1I Alignment of monkeypox virus B21R gene DNA (bases 1967-2216) and other orthopoxvirus homologous gene sequences;

[0047] Figure 1J Alignment of monkeypox virus B21R gene DNA (bases 2217-2516) and other orthopoxvirus homologous gene sequences;

[0048] Figure 1K Alignment of monkeypox virus B21R gene DNA (bases 2517-2766) and other orthopoxvirus homologous gene sequences;

[0049] Figure 1L Alignment of monkeypox virus B21R gene DNA (bases 2767-3016) and other orthopoxvirus homologous gene sequences;

[0050] Figure 1M Alignment of monkeypox virus B21R gene DNA (bases 3017-3316) and other orthopoxvirus homologous gene sequences;

[0051] Figure 1N Alignment of monkeypox virus B21R gene DNA (bases 3317-3566) and other orthopoxvirus homologous gene sequences;

[0052] Figure 1O Alignment of monkeypox virus B21R gene DNA (bases 3567-3816) and other orthopoxvirus homologous gene sequences;

[0053] Figure 1P Alignment of monkeypox virus B21R gene DNA (bases 3817-4116) and other orthopoxvirus homologous gene sequences;

[0054] Figure 1Q Alignment of monkeypox virus B21R gene DNA (bases 4117-4366) and other orthopoxvirus homologous gene sequences;

[0055] Figure 1RComparison of monkeypox virus B21R gene DNA (4367-4616 bases) and other similar orthopoxvirus homologous gene sequences;

[0056] Figure 1S Comparison of monkeypox virus B21R gene DNA (4617-4916 bases) and other similar orthopoxvirus homologous gene sequences;

[0057] Figure 1T Comparison of monkeypox virus B21R gene DNA (4917-5166 bases) and other similar orthopoxvirus homologous gene sequences;

[0058] Figure 1U Comparison of monkeypox virus B21R gene DNA (5167-5416 bases) and other similar orthopoxvirus homologous gene sequences;

[0059] Figure 1V Comparison of monkeypox virus B21R gene DNA (5417-5643 bases) and other similar orthopoxvirus homologous gene sequences;

[0060] Figure 2 Conserved B21R amplicon sequence in monkeypox virus genomes of different years;

[0061] Figure 3 Amplification curve when the forward primer is SEQ ID NO. 1, the reverse primer is SEQ ID NO. 2, and the fluorescent probe is SEQ ID NO. 5;

[0062] Figure 4 Amplification curve when the forward primer is SEQ ID NO. 3, the reverse primer is SEQ ID NO. 4, and the fluorescent probe is SEQ ID NO. 6;

[0063] Figure 5 Standard curve plot according to Figure 3 the results;

[0064] Figure 6 Standard curve plot according to Figure 4 the results;

[0065] Figure 7The multiplex PCR amplification curve chart when the forward primer 1 is SEQ ID NO. 1, the reverse primer 1 is SEQ ID NO. 2, the fluorescent probe 1 is SEQ ID NO. 5, the forward primer 2 is SEQ ID NO. 7, the reverse primer 2 is SEQ ID NO. 8, the fluorescent probe 2 is SEQ ID NO. 9 (5' end with FAM fluorescent group, 3' end with BHQ1 quenching group), the forward primer 3 is SEQ ID NO. 10, the reverse primer 3 is SEQ ID NO. 11, and the fluorescent probe 3 is SEQ ID NO. 12 (5' end with CY5 fluorescent group, 3' end with BHQ2 quenching group);

[0066] Figure 8 The multiplex PCR amplification curve chart when the forward primer 1 is SEQ ID NO. 3, the reverse primer 1 is SEQ ID NO. 4, the fluorescent probe 1 is SEQ ID NO. 6, the forward primer 2 is SEQ ID NO. 7, the reverse primer 2 is SEQ ID NO. 8, the fluorescent probe 2 is SEQ ID NO. 9 (5' end with FAM fluorescent group, 3' end with BHQ1 quenching group), the forward primer 3 is SEQ ID NO. 10, the reverse primer 3 is SEQ ID NO. 11, and the fluorescent probe 3 is SEQ ID NO. 12 (5' end with CY5 fluorescent group, 3' end with BHQ2 quenching group);

[0067] Figure 9 The multiplex PCR amplification curve chart when the forward primer is SEQ ID NO. 1, the reverse primer is SEQ ID NO. 2, the fluorescent probe is SEQ ID NO. 5, and the fragment containing the camel pox B21R gene is the template (SEQ ID NO. 15);

[0068] Figure 10 The multiplex PCR amplification curve chart when the forward primer is SEQ ID NO. 3, the reverse primer is SEQ ID NO. 4, the fluorescent probe is SEQ ID NO. 6, and the fragment containing the camel pox B21R gene is the template (SEQ ID NO. 15). DETAILED DESCRIPTION

[0069] In order to further illustrate the technical means adopted by the present application and its effects, the present application will be further described below in conjunction with the embodiments and drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, and not to limit the present application.

[0070] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0071] Example 1

[0072] Identification of specific B21R target genes.

[0073] (1) The highly specific target gene B21R was selected from the whole genome sequence of monkeypox virus using multiple DNA sequence comparison. Monkeypox virus-specific primers and probes are underlined, and amplicon is marked with a box. Figure 1I -J), by Figures 1A-1V The gene sequence comparison results show that the B21R gene is polymorphic in various orthopoxviruses, from which monkeypoxvirus-specific gene fragments can be found as targets for fluorescent PCR.

[0074] (2) Based on multiplex DNA sequence comparison, two pairs of highly specific primers and two corresponding fluorescent probes were designed. Figures 1A-1V Sequence comparison of the amplicon shows that the newly designed B21R target site does not exist in other related orthopoxviruses except for camelpox. The allele sequence fragment of camelpox is also quite different from the target site of the designed monkeypox B21R amplicon, so theoretically it will not be amplified by the designed primers and the fluorescent signal will not be detected.

[0075] (3) From Figure 2 Comparison of monkeypox virus genome sequences collected from different years shows that the selected target sites are highly conserved, with no high-frequency mutations and only one repetitive insertion of TAC, but this does not affect the binding of the designed primers and will not produce off-target effects. Further Blast search confirms the specificity of the designed primers.

[0076] (4) The corresponding camelpox DNA fragment was synthesized and amplified using specific monkeypox primers and probes. No amplification was observed, thus identifying two specific target sites on B21R.

[0077] Example 2

[0078] Fluorescent PCR detection of the B21R target site based on Example 1.

[0079] The primers, fluorescent probes, and plasmids containing the monkeypox virus target gene (B21R) fragment were obtained by chemical synthesis according to the DNA sequence. The plasmid containing the monkeypox virus target gene (B21R) fragment was used as the amplification template (SEQ 13), and the target gene concentration was 10 pg, 1 pg, 0.1 pg, 0.01 pg, 0.001 pg. The reaction system was prepared according to Table 1. The PCR amplification program was as follows: 95 °C, 10 min, 1 cycle; 95 °C, 30 s; 60 °C, 1 min, 40 cycles. The PCR amplification and detection were performed on an ABI 7500 Real-Time PCR instrument (Thermo). When the forward primer was SEQ ID NO. 1, the reverse primer was SEQ ID NO. 2, and the fluorescent probe was SEQ ID NO. 5, the amplification curve was as shown in Figure 3 , and the standard curve was as shown in Figure 5 .

[0080] SEQ ID NO. 13:

[0081]

[0082] When the target gene concentration is 10 pg, 1 pg, 0.1 pg, 0.01 pg, 0.001 pg, the reaction system is prepared according to Table 2, the PCR amplification procedure is: 95°C, 10 min, 1 cycle; 95°C, 30 s; 60°C, 1 min, 40 cycles, when the forward primer is SEQ ID NO. 3, the reverse primer is SEQ ID NO. 4, and the fluorescent probe is SEQ ID NO. 6, the amplification curve is as shown in Figure 4 , and the standard curve is as shown in Figure 6 .

[0083] Table 1

[0084] Component Amount AceQ qPCR Probe Master Mix 10 μL Forward primer (10 μM) 0.4 μL Reverse primer (10 μM) 0.4 μL Fluorescent probe (10 μM) 0.4 μL ddH2O 7.8 μL Template 1 μL

[0085] Results: As shown in Figure 4 and Figure 5 , the two pairs of primers and probes designed can better detect the monkeypox B21R target site, and the amplification curve is good, as shown in Figure 6 and Figure 7 , within the range of 0.001 pg to 100 pg of the template concentration, the standard curve is linear (triplicate, R 2 > 0.99, and the amplification efficiency (100%-102%) is good; at the same time, the camelpox gene (template concentration up to 10 pg) cannot be detected, indicating that the amplification primer and probe system used has good specificity.

[0086] Example 3

[0087] Multiplex fluorescent PCR detection.

[0088] The amplification target of the primer is the B21R gene, J2R gene (SEQ ID NO. 14) and the internal reference gene (human RNaseP gene) of the monkeypox virus, respectively. The primers, fluorescent probes and plasmids containing target gene fragments are obtained by chemical synthesis according to the DNA sequence, the reaction system is prepared according to Table 2, the PCR amplification procedure is: 95°C, 10 min, 1 cycle; 95°C, 30 s; 60°C, 1 min, 40 cycles, and the PCR amplification and detection are performed on an ABI 7500 Real-Time PCR instrument (Thermo).

[0089] SEQ ID NO. 14:

[0090]

[0091] The multiplex PCR amplification curve is shown in FIG. 1 when forward primer 1 is SEQ ID NO. 1, reverse primer 1 is SEQ ID NO. 2, fluorescent probe 1 is SEQ ID NO. 5, forward primer 2 is SEQ ID NO. 7, reverse primer 2 is SEQ ID NO. 8, fluorescent probe 2 is SEQ ID NO. 9 (5' end with FAM fluorescent group, 3' end with BHQ1 quenching group), forward primer 3 is SEQ ID NO. 10, reverse primer 3 is SEQ ID NO. 11, and fluorescent probe 3 is SEQ ID NO. 12 (5' end with CY5 fluorescent group, 3' end with BHQ2 quenching group). Figure 7

[0092] SEQ ID NO. 7: GGAAAATGTAAAGACAACGAATACAG.

[0093] SEQ ID NO. 8: GCTATCACATAATCTGGAAGCGTA.

[0094] SEQ ID NO. 9: AAGCCGTAATCTATGTTGTCTATCGTGTCC.

[0095] SEQ ID NO. 10: AGATTTGGACCTGCGAGCG.

[0096] SEQ ID NO. 11: GAGCGGCTGTCTCCACAAGT.

[0097] SEQ ID NO. 12: TTCTGACCTGAAGGCTCTGCGCG.

[0098] The multiplex PCR amplification curve is shown in FIG. 2 when forward primer 1 is SEQ ID NO. 3, reverse primer 1 is SEQ ID NO. 4, fluorescent probe 1 is SEQ ID NO. 5, forward primer 2 is SEQ ID NO. 7, reverse primer 2 is SEQ ID NO. 8, fluorescent probe 2 is SEQ ID NO. 9 (5' end with FAM fluorescent group, 3' end with BHQ1 quenching group), forward primer 3 is SEQ ID NO. 10, reverse primer 3 is SEQ ID NO. 11, and fluorescent probe 3 is SEQ ID NO. 12 (5' end with CY5 fluorescent group, 3' end with BHQ2 quenching group). Figure 8

[0099] ​​The forward primer is SEQ ID NO. 1, the reverse primer is SEQ ID NO. 2, the fluorescent probe is SEQ ID NO. 5, and the fragment containing the B21R gene of the Lao camel pox is the template (SEQ ID NO. 15), and the amplification curve is as shown in Figure 9 .

[0100] SEQ ID NO. 15:

[0101]

[0102] The forward primer is SEQ ID NO. 3, the reverse primer is SEQ ID NO. 4, the fluorescent probe is SEQ ID NO. 6, and the fragment containing the B21R gene of the camelpox is the template (SEQ ID NO. 15), and the amplification curve is as shown in Figure 10 .

[0103] Table 2

[0104] Component Amount AceQ qPCR Probe Master Mix 10 μL Forward primer 1 (10 μM) 0.4 μL Reverse primer 1 (10 μM) 0.4 μL Fluorescent probe 1 (10 μM) 0.4 μL Forward primer 2 (10 μM) 0.4 μL Reverse primer 2 (10 μM) 0.4 μL Fluorescent probe 2 (10 μM) 0.4 μL Forward primer 3 (10 μM) 0.4 μL Reverse primer 3 (10 μM) 0.4 μL Fluorescent probe 3 (10 μM) 0.4 μL ddH2O 5.4 μL Template 1 μL

[0105] Results: As shown in Figure 7 and Figure 8 , the amplification curves of the target points B21R and J2R of the two monkeypox viruses are good, and are all detected, but the camel pox gene fragment cannot be detected ( Figure 9 and Figure 10 ), and all the internal reference genes can be normally amplified, so the multiplex PCR method for detecting monkeypox virus of the present application has sensitivity and specificity, is less likely to produce off-target effects than the original method, and the specificity is further enhanced.

[0106] In summary, the present application creatively designs two pairs of primers and corresponding fluorescent probes, which can specifically detect the new target gene B21R in the monkeypox virus DNA, and the target site has conservation and specificity; in addition, a duplex fluorescent PCR method is established, which can further improve the specificity and accuracy of the detection of monkeypox virus.

[0107] The applicant declares that the present application is illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed methods, that is, it does not mean that the present application must rely on the above-mentioned detailed methods to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A primer and a fluorescent probe for detection of monkeypox virus, characterized by, The nucleic acid sequence of the primers for detecting the monkeypox virus comprises the sequences shown in SEQ ID NO. 1-SEQ ID NO. 4; the nucleic acid sequence of the fluorescent probe for detecting the monkeypox virus comprises the sequences shown in SEQ ID NO. 5-SEQ ID NO.

6.

2. The primer and fluorescent probe for detection of monkeypox virus according to claim 1, characterized by, The 5' end of the fluorescent probe contains a fluorescent group, and the 3' end contains a quenching group. 3.The primer and fluorescent probe for detection of monkeypox virus according to claim 2, characterized in that, The fluorescent group comprises any one of VIC, FAM, or CY5, or a combination of at least two thereof. 4.The primer and fluorescent probe for detection of monkeypox virus according to claim 2, characterized in that, The quenching group comprises BHQ1 and / or BHQ2.

5. Use of the primers and fluorescent probe for detecting the monkeypox virus according to claim 1 in the preparation of a product for detecting the monkeypox virus.

6. A kit for detection of monkeypox virus, characterized in that, The kit comprises the primers and fluorescent probe for detecting the monkeypox virus as described in claim 1.

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