Primer group for detecting mumps virus, application of primer group, kit and application of kit

Abstract

The invention relates to the technical field of chemical synthesis and diagnostic reagents, in particular to a primer group for detecting mumps virus, application of the primer group, a kit and application of the kit. The mumps virus NCRs multiplex RT-PCR amplification method is established on the basis of a multiplex PCR principle, one tube for three detections is realized, amplification products of three non-coding regions of N-P, P-M and M-F are obtained through successful amplification, and a full-length sequence of the NCRs is obtained through sequence splicing. Based on the primer group, the invention provides the primer group for detecting the mumps virus NCRs. A gene sequence of mumps virus NCRs is analyzed by a bioinformatics method, and three groups of primers respectively aiming at three non-coding regions of N-P, P-M and M-F are synthesized by a chemical synthesis technology. The primer group provided by the invention can be used for identifying the MuV, can also be used for simultaneously detecting three non-coding region sequences of N-P, P-M and M-F of the MuV, and is used for molecular virology monitoring.

Description

Technical Field

[0001] This application relates to the technical field of chemical synthesis and diagnostic reagents, and in particular to a primer set and its application for detecting mumps virus, a reagent kit and its application. Background Technology

[0002] Mumps (epidemic parotitis) is an acute respiratory infectious disease caused by the mumps virus (MuV). Clinically, it mainly manifests as unilateral / bilateral non-suppurative swelling and pain of the parotid glands. It can also lead to complications such as pancreatitis, orchitis, deafness, aseptic meningitis, and encephalitis. Mumps meningitis is one of the main and most common complications, with approximately 10% of MuV infections developing aseptic meningitis. Clinical symptoms mainly include fever, vomiting, headache, and neck stiffness. Currently, MuV can be classified into 12 genotypes globally, named AN (excluding E and M). Different MuV genotypes exhibit significant geographical distribution; genotypes C, G, H, J, and K are mainly prevalent in the Western Hemisphere, while genotypes B, F, I, and L are mainly found in Asia. Since 2010, only 6 of the 12 MuV genotypes have become prevalent in 25 countries: genotypes G, H, C, F, K, and D.

[0003] Since the introduction of MuV live attenuated vaccine in the 1960s, the widespread adoption of MuCV has significantly reduced the global incidence of mumps. In countries implementing two-dose MMR vaccination, the incidence of mumps has plummeted from 100-1000 cases per 100,000 people to <1 case per 100,000 people, confirming the protective effect of MuCV against MuV infection. However, recently, large-scale outbreaks of mumps caused by G genotype MuV have frequently occurred in countries with high vaccination coverage, such as the United States, Canada, the United Kingdom, and France. A large number of identical SH gene sequences have been detected during these outbreaks, and the limited diversity of SH genes has hindered effective virus tracing and transmission chain identification in MuV molecular epidemiological investigations. In my country, the predominantly circulating strain is F genotype MuV. Studies have shown that F genotype MuV in my country is gradually shifting towards a single dominant circulating strain, with continuously decreasing SH gene genetic diversity. Using only SH gene typing for etiological surveillance of F genotype MuV in my country has revealed numerous problems, including low resolution and difficulty in virus tracing.

[0004] Whole genome sequencing (WGS) is a powerful and crucial technique for studying the transmission patterns of MuV strains. However, currently only a few countries and teams are capable of performing MuV whole genome sequencing. High sample quality requirements, expensive testing costs, and high technical barriers also limit the application of WGS in current MuV molecular epidemiology research. Target gene sequencing technology remains the most common method in MuV molecular epidemiology research. Currently, the SH gene is the most commonly used molecular target for MuV molecular epidemiological surveillance. Meanwhile, the HN and F genes are also frequently used for MuV genotyping and molecular source tracing analysis. However, because the HN and F genes are important functional protein-coding genes in MuV, their gene sequence fragments are long and highly conserved, thus their application in MuV molecular epidemiology research is very limited.

[0005] Previous studies have shown that the three non-coding regions (NCRs) located between the N and P genes (NP), between the P and M genes (PM), and between the M and F genes (MF) exhibit the greatest variability across the entire genome. Some studies have also attempted to use NCRs as supplementary molecular evidence for SH gene tracing to improve the resolution of MuV molecular epidemiology. However, global research data on NCRs remains limited, and a primer set suitable for amplifying F genotype MuV NCR sequences in my country is still lacking. Summary of the Invention

[0006] This application provides a primer set and its application for detecting mumps virus, as well as a reagent kit and its application.

[0007] To address the above issues, this application provides a multiplex RT-PCR amplification technique for non-coding regions (NCRs) of mumps virus, replacing the SH gene for MuV tracing and transmission chain identification, improving the molecular virological surveillance capability of F genotype MuV, revealing the potential transmission patterns of the F genotype mumps virus in my country, and providing a new technical means for mumps pathogen surveillance in my country and globally. NCRs as molecular markers for virological surveillance of the F genotype mumps virus have certain feasibility and practical value.

[0008] Based on the principle of multiplex PCR, this application establishes a multiplex RT-PCR amplification method for mumps virus NCRs, achieving three detections in one tube. Simultaneously, it successfully amplifies the amplification products of the three non-coding regions NP, PM and MF, and obtains the full-length NCRs sequence through sequence splicing.

[0009] Based on the above, this application provides a primer set for detecting mumps virus NCRs. The gene sequences of mumps virus NCRs were analyzed using bioinformatics methods, and three sets of primers targeting the NP, PM, and MF non-coding regions were synthesized using chemical synthesis techniques. The primer set provided in this application can be used to identify MuV and simultaneously detect the NP, PM, and MF non-coding region sequences of MuV, for use in molecular virological surveillance.

[0010] In a first aspect, this application provides a primer set for detecting mumps virus, employing the following technical solution:

[0011] A primer set for detecting mumps virus, the primer set being used to amplify the non-coding region of the mumps virus; the non-coding region comprises three parts, namely NP, PM and MF;

[0012] The primer set used to amplify NP includes the nucleotide sequence as described in SEQ ID No. 1 and the nucleotide sequence as described in SEQ ID No. 2;

[0013] The primer set used to amplify PM includes the nucleotide sequence as described in SEQ ID No. 3 and the nucleotide sequence as described in SEQ ID No. 4;

[0014] The primer set used to amplify MF includes the nucleotide sequences described in SEQ ID No. 5 and SEQ ID No. 6.

[0015] Multiplex PCR can increase the number of amplified fragments by optimizing the reaction system and conditions. It combines the specificity and sensitivity of single PCR with the advantages of speed and economy, and has been widely used in many fields, including nucleic acid diagnostics, since its inception. However, the difficulty in designing multiplex PCR lies in the incompatibility of amplification conditions between multiple targets and the easy interference between primers for amplification of different targets, resulting in poor amplification effect in the actual operation of multiplex PCR.

[0016] In multiplex PCR experiments, the primer pairs used must not bind to each other or to regions of the template DNA other than the target fragment. Furthermore, in traditional multiplex PCR experiments, to facilitate gel detection, different amplicon fragments of varying sizes must be designed. Adding to the complexity, different amplified fragments in multiplex PCR compete for resources, resulting in high-abundance templates being easily detected while low-abundance templates become mere background noise. Therefore, in addition to following general rules, primer design should also consider the following points:

[0017] a. Primer specificity: Each primer should not have significant complementarity with other amplification fragments, nor should the amplification fragments have significant homology with each other;

[0018] b. Primer length: Generally 18-24 bases. Primers should not be complementary, especially avoiding 3' end complementarity. Longer primers are more likely to form dimers;

[0019] c. Annealing temperature: Selection criteria for annealing temperature: Increase the annealing temperature of each primer pair, and then use the lowest annealing temperature during mPCR;

[0020] d. Primer structure design: Unique structural designs can be used to avoid non-specific amplification or primer dimers. For example, the Ω primer consists of three parts: a 5' end sequence, an Ω loop, and a 3' end sequence. The two end sequences are complementary to the target region, while the Ω loop is not. For the Ω primer, the target region can only be amplified when the 5' and 3' end sequences are perfectly paired; otherwise, its structure is unstable (due to the presence of the Ω loop), leading to amplification failure.

[0021] Multiplex PCR requires specific amplification of multiple sites within the same reaction system; therefore, primer pairing and competitive amplification can both affect the amplification efficiency. Choosing appropriate reaction systems and conditions can improve the amplification effect of multiplex PCR. Therefore, when performing multiplex PCR, one should not rigidly adhere to conventional methods but continuously optimize experimental conditions based on one's own experimental circumstances to achieve the best amplification results.

[0022] Based on the above, this application used bioinformatics technology and experimental verification to finally screen out multiplex PCR primer sets that have good amplification efficiency for A, F, and G genotypes, and screened out the most suitable reaction conditions, thus establishing a complete and efficient multiplex PCR amplification technology for mumps virus NCRs.

[0023] Secondly, this application provides a kit for detecting mumps virus. The kit includes the aforementioned primer set. The kit also includes at least one of RT-PCR amplification reagents and PCR amplification reagents.

[0024] Thirdly, this application provides the use of the above-mentioned primer set or kit in the preparation of products for detecting mumps virus.

[0025] In summary, this application includes at least one of the following beneficial technical effects:

[0026] The primer set provided in this application can be used to identify MuV and can also simultaneously detect the NP, PM and MF non-coding regions of MuV for molecular virology surveillance. Attached Figure Description

[0027] Figure 1 The electrophoresis results are those of the MuV NP genotype amplified using three sets of primers in Example 2.

[0028] Figure 2 The electrophoresis results are those of the MuV NM genotype amplified using three sets of primers in Example 2.

[0029] Figure 3 The electrophoresis results are those of the MuV NF genotype amplified using three sets of primers in Example 2.

[0030] Figure 4 The electrophoresis results are from the multiple detection system test of the first set of multiple primers in Example 3.

[0031] Figure 5 The electrophoresis results are from the multiple detection system test of the second set of multiple primers in Example 3.

[0032] Figure 6 The electrophoresis results are from the multiplex detection system test of genotype A MuV using the first and second primer sets in Example 4.

[0033] Figure 7 The electrophoresis results are from the multiplex detection system test of G genotype MuV using the first and second primer sets in Example 4. Detailed Implementation

[0034] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0035] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0036] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0037] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0038] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0039] 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 commercially available conventional products.

[0040] The present application will be further described in detail below with reference to the embodiments and test results.

[0041] Example

[0042] Example 1

[0043] This embodiment provides the design and synthesis of primers.

[0044] The sequences of the three non-coding regions of mumps virus were obtained from the NCBI nucleotide database as follows: the NP sequence is as described in SEQ ID No. 19; the PM sequence is as described in SEQ ID No. 20; and the MF sequence is as described in SEQ ID No. 21 (obtained by cutting the whole genome of F genotype MuVs, Genbank: MT350231).

[0045] To ensure effective differentiation of multiplex PCR primers under gel electrophoresis, upstream and downstream primers for the three non-coding regions were designed using the bioinformatics software MEGA-X with standard nucleotide lengths of 200 bp (PM), 400 bp (NP), and 600 bp (MF), respectively. Structural analysis was performed using Primer Premier 5 software. Primer specificity and matching analysis were conducted using NCBI's Primer Blast function to ensure high specificity for the three non-coding regions and to prevent degeneracy that could lead to missed detections. Finally, the primers were screened under multiplex amplification conditions using MFEprimer software to avoid non-specific amplification or primer dimers between multiplex primer sets and to ensure similar annealing temperatures among primer sets. The final three sets of primers for each of the three non-coding regions, with specific nucleotide sequences (5'-3'), are shown in Table 1.

[0046] Table 1 shows three sets of primers for each of the three non-coding regions.

[0047]

[0048]

[0049] Example 2

[0050] This embodiment uses the primers from Example 1 for single-weight testing.

[0051] PrimeScript sold by TAKARA TM The One Step RT-PCR Kit Ver.2 (Dye Plus) is used as the amplification reaction system.

[0052] Three sets of primers for each of the three non-coding regions of mumps virus were tested in single-weight systems.

[0053] The amplification system is shown in Table 2.

[0054] Table 2 Amplification System 1

[0055] Serial Number content Amount of 1 sample 1 Primer Script 1step Enzyme Mix 1ul 2 2X1 Step Buffer 12.5ul 3 Upstream primer: (20 μM) 1ul 4 Downstream primer: (20 μM) 1ul 5 <![CDATA[H2O]]> 4.5ul 6 RNA template 5ul

[0056] The amplification program settings are shown in Table 3.

[0057] Table 3 Amplification Procedure 1

[0058]

[0059] The amplification data of the three sets of primers for each of the three non-coding regions of mumps virus are shown in Tables 4-6 below (F genotype). The amplification results are as follows: Figure 1-3 As shown.

[0060] Table 4. Amplification results of F genotype MuV NP

[0061]

[0062]

[0063] Table 5. Amplification results of F genotype MuV PM

[0064] Virus dilution CT value PM(F1,R1) PM(F2,R2) PM(F3,R3) <![CDATA[1×10 -1 ]]> 13.952 + + + <![CDATA[1×10 -2 ]]> 17.135 + + + <![CDATA[1×10 -3 ]]> 21.530 + + + <![CDATA[1×10 -4 ]]> 24.542 + - - <![CDATA[1×10 -5 ]]> 27.475 - - -

[0065] Table 6. Amplification results of MuV MF with genotype F

[0066] Virus dilution CT value MF(F1,R1) MF(F2,R2) MF(F3,R3) <![CDATA[1×10 -1 ]]> 13.952 + + + <![CDATA[1×10 -2 ]]> 17.135 + + - <![CDATA[1×10 -3 ]]> 21.530 + + - <![CDATA[1×10 -4 ]]> 24.542 + + - <![CDATA[1×10 -5 ]]> 27.475 + + -

[0067] From Tables 2-4 and Figure 1-3 The amplification results showed that the first and second primer sets had good amplification effects on all three target gene segments, while the third primer set could not successfully amplify the NP and MF sequences. Therefore, the first and second primer sets were selected to further investigate the multiplex amplification effect.

[0068] Example 3

[0069] Based on the results of Example 2, this example uses the first and second sets of primers from Example 1 to test the multiplex detection system for F genotype MuV.

[0070] PrimeScript sold by TAKARA TM The One Step RT-PCR Kit Ver.2 (Dye Plus) is used as the amplification reaction system.

[0071] A multiplex detection system was established using three sets of primers for each of the three non-coding regions of the mumps virus.

[0072] The amplification system is shown in Table 7.

[0073] Table 7 Amplification System II

[0074]

[0075]

[0076] The amplification program settings are shown in Table 8.

[0077] Table 8 Amplification Procedure 2

[0078]

[0079] (1) First, the first set of multiplex primers was selected for testing of the multiplex detection system (F genotype MuV). The amplification data are shown in Table 9 below, and the electrophoresis results are as follows. Figure 4 As shown.

[0080] Table 9. Multiplex Detection System – First Set of Alternative Primers

[0081] Virus dilution CT value NP(F1,R1) PM(F1,R1) MF(F1,R1) <![CDATA[1×10 -1 ]]> 13.952 + + + <![CDATA[1×10 -2 ]]> 17.135 + + + <![CDATA[1×10 -3 ]]> 21.530 + + + <![CDATA[1×10 -4 ]]> 24.542 + + + <![CDATA[1×10 -5 ]]> 27.475 + + +

[0082] Based on the test results in Table 9 above and Figure 4 It can be seen that the amplified bands are clear, and the amplified bands of different lengths are distinct. The first set of candidate primers has a good amplification effect on the target genes of F genotype MuV three-segment NCRs and has high amplification sensitivity.

[0083] (2) Next, a second set of multiplex primers was selected for testing the multiplex detection system (F genotype MuV). The amplification data are shown in Table 10 below. Electrophoresis results are as follows. Figure 4 As shown.

[0084] Table 10. Multiple Detection System – Second Set of Alternative Primers

[0085] Virus dilution CT value NP(F2,R2) PM(F2,R2) MF(F2,R2) <![CDATA[1×10 -1 ]]> 13.952 + + + <![CDATA[1×10 -2 ]]> 17.135 + + + <![CDATA[1×10 -3 ]]> 21.530 + + + <![CDATA[1×10 -4 ]]> 24.542 + + + <![CDATA[1×10 -5 ]]> 27.475 + + +

[0086] Based on the test results in Table 10 above and Figure 5 It can be seen that the amplified bands are clear, and the amplified bands of different lengths are distinct. The second set of candidate primers has a good amplification effect on the target genes of F genotype MuV three-segment NCRs and has high amplification sensitivity.

[0087] As shown in Table 9-10, both the first and second sets of candidate primers have good amplification efficiency and high amplification sensitivity for F genotype MuV.

[0088] Example 4

[0089] This embodiment tested the universality of MuV for genes A and G.

[0090] PrimeScript sold by TAKARA TM The One Step RT-PCR Kit Ver.2 (Dye Plus) is used as the amplification reaction system.

[0091] The amplification system is shown in Table 11.

[0092] Table 11 Amplification System Three

[0093] Serial Number content Amount of 1 sample 1 Primer Script 1 step Enzyme Mix 1ul 2 2X1 Step Buffer 12.5ul 3 F / R primer NV (5μM) (bidirectional primer) 1.5ul 4 F / R primer PM (5μM) (bidirectional primer) 1ul 5 F / R primer MF (5μM) (bidirectional primer) 0.5 6 <![CDATA[H2O]]> 3.5ul 7 RNA template 2ul

[0094] The amplification program settings are shown in Table 12.

[0095] Table 12 Amplification Program 3

[0096]

[0097] (1) Genotype A

[0098] The first and second primer sets were used to detect MuV genotype A, and their specificity and sensitivity were investigated. Data are shown in Tables 13-14. Electrophoresis results are as follows: Figure 6 As shown.

[0099] Table 13 First set of primers - Genotype A MuV

[0100]

[0101]

[0102] Table 14 Second set of primers - Genotype A MuV

[0103] Virus dilution CT value NP(F1,R1) PM(F1,R1) MF(F1,R1) <![CDATA[1×10 -1 ]]> 24.529 - - + <![CDATA[1×10 -2 ]]> 28.261 - - + <![CDATA[1×10 -3 ]]> 33.263 - - + <![CDATA[1×10 -4 ]]> 37.411 - - - <![CDATA[1×10 -5 ]]> None - - -

[0104] (2) G genotype

[0105] The first and second primer sets were used to detect MuV of the G genotype, and their specificity and sensitivity were investigated. Data are shown in Tables 15-16. Electrophoresis results are as follows. Figure 7 As shown.

[0106] Table 15 First set of primers - G genotype MuV

[0107] Virus dilution CT value NP(F1,R1) PM(F1,R1) MF(F1,R1) <![CDATA[1×10 -1 ]]> 15.333 + + + <![CDATA[1×10 -2 ]]> 18.739 + + + <![CDATA[1×10 -3 ]]> 22.972 + + + <![CDATA[1×10 -4 ]]> 26.323 + + + <![CDATA[1×10 -5 ]]> 28.386 - - -

[0108] Table 16 Second set of primers - G genotype MuV

[0109] Virus dilution CT value NP(F1,R1) PM(F1,R1) MF(F1,R1) <![CDATA[1×10 -1 ]]> 15.333 + + - <![CDATA[1×10 -2 ]]> 18.739 + + - <![CDATA[1×10 -3 ]]> 22.972 + + - <![CDATA[1×10 -4 ]]> 26.323 + + - <![CDATA[1×10 -5 ]]> 28.386 - - -

[0110] From Table 15-16 and Figures 6-7 The amplification results showed that the first set of candidate primers had good amplification effects on both genotype A and genotype G MuV, while the second set of candidate primers could not successfully amplify the NP and PM fragments of genotype A and the MF fragment of genotype G.

[0111] Therefore, the first set of candidate primers was ultimately chosen as the final multiplex PCR primer set.

[0112] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A primer set for detecting mumps virus, characterized in that, The primer set is used to amplify the non-coding region of the mumps virus; the non-coding region consists of three parts, namely NP, PM and MF; The primer set for amplifying NP includes the nucleotide sequences as described in SEQ ID No. 1 and the nucleotide sequences as described in SEQ ID No. 2; The primer set used to amplify PM includes the nucleotide sequence as described in SEQ ID No. 3 and the nucleotide sequence as described in SEQ ID No. 4; The primer set used to amplify MF includes the nucleotide sequences described in SEQ ID No. 5 and SEQ ID No.

6.

2. A kit for detecting mumps virus, characterized in that, The kit includes the primer set as described in claim 1.

3. The reagent kit according to claim 1, characterized in that, The kit also includes at least one of RT-PCR amplification reagents and PCR amplification reagents.

4. Use of the primer set of claim 1 or the kit of any one of claims 2-3 in the preparation of a product for detecting mumps virus.

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