A primer set, probe and kit and their applications

Abstract

The present application relates to the field of biological detection technology, and more particularly to a primer set, a probe, a kit, and applications thereof, wherein the primer set includes an amplification primer pair. The sequence of the amplification primer pair includes an upstream primer and a downstream primer. The primer set and the probe are used to detect Epstein-Barr virus.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a primer set, a probe, a kit and applications thereof. Background Art

[0002] Epstein-Barr virus (EBV) is a herpesvirus type IV in the family Herpesviridae. It is a herpesvirus that is tropic for human lymphocytes. EBV infection is highly prevalent in the human population, with over 90% of patients testing positive for serum EBV antibodies. In addition to primary EBV infection causing infectious mononucleosis, it can also cause severe non-neoplastic EBV-related diseases such as chronic active EBV infection and EBV-associated hemophagocytic lymphohistiocytosis. EBV is also an oncogenic virus, implicated in the development of numerous tumors, including Hodgkin's lymphoma, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, gastric cancer, and post-transplant lymphoproliferative disorder. Therefore, it is crucial to adopt appropriate diagnosis and treatment strategies for each EBV-related disease.

[0003] Currently, the main laboratory diagnostic methods for EBV infection include EBV-specific antibody testing, heterophilic agglutination antibody testing, EBV nucleic acid load testing, and EBERs in situ hybridization testing. Recent research results indicate that serum / plasma EBV-DNA can be used for nasopharyngeal carcinoma screening, particularly for asymptomatic early-stage NPC patients, and may be more sensitive than the VCA-IgA assay. Furthermore, plasma EBV-DNA can be used for post-treatment monitoring and prognosis assessment of NPC patients. Furthermore, EBV-DNA nucleic acid load testing can serve as an important marker of tumor burden in patients with EBV-related lymphomas and can be used to assess treatment efficacy and predict prognosis.

[0004] Existing EBV-DNA detection is mainly based on real-time fluorescence PCR technology. That is, based on the working principle of Taqman probe, each round of amplification accumulates a fluorescent signal, which is used to monitor the entire PCR process in real time. Finally, the unknown template is qualitatively or quantitatively analyzed through the standard curve. However, there are problems with long detection time and cumbersome operation. Summary of the Invention

[0005] The present invention provides a primer set, a probe, a kit, and their use. The method utilizes the kit to detect Epstein-Barr virus. The method has a short detection time and is simple to operate.

[0006] To achieve the above objectives, the embodiments of the present application adopt the following technical solutions:

[0007] In a first aspect, embodiments of the present application provide a primer set and a probe. The primer set includes an amplification primer pair. The sequence of the amplification primer pair includes an upstream primer and a downstream primer.

[0008] Upstream primer: 5′-CCTCACATACACCTTACTGTTCACAACTCAGC-3′.

[0009] Downstream primer: 5′-GTCCTAAGGAACAGCGATATCTCCCACCCCAT-3′.

[0010] The probe sequences include:

[0011] 5'-GAAGAAGCAGGCGAAGATTCAGGAGAGTTCACTGCCCGCTCCTTGA-3'.

[0012] The primer set and probe provided in the embodiments of the present application can realize simultaneous amplification and detection without the need to open the lid, and can realize primer amplification and result detection in a fully closed state, overcoming the problem of existing primer sets that require an amplification reaction to be performed first during detection, and then the lid needs to be opened after the reaction results are obtained to transfer the results to the detection platform for detection and analysis. Compared with existing detection, the detection time is shortened. In addition, without the need to open the lid, the integrated operation process of amplification and detection is realized, which also avoids environmental contamination and improves detection readiness.

[0013] In some embodiments, the probe is labeled with a fluorescent group, a quencher group, and THF, the fluorescent group and the quencher group are both labeled on T bases, the fluorescent group and the quencher group are separated by 2 bp to 6 bp, the THF is located between the fluorescent group and the quencher group, the THF is ≥30 bp away from the 5' end of the probe, the THF is ≥15 bp away from the 3' end of the probe, and the THF is located between adjacent T bases.

[0014] In some embodiments, the fluorescent group includes FAM, TET, HEX, CY3, or JOE, and the quencher group includes BHQ1, BHQ2, TAMRA, DABCYL, MGB, or Eclipse.

[0015] In some embodiments, the 3' end of the probe is further labeled with a blocking group.

[0016] In some embodiments, the blocking group comprises an amine group, a phosphate group, biotin-TEG, a sulfhydryl group, or a C3-Spacer.

[0017] In a second aspect, the present invention also provides a kit comprising the above-mentioned primer set and probe.

[0018] The method of the embodiment of the present application can achieve the same technical effect as the kit provided in the above embodiment, and will not be repeated here.

[0019] In some embodiments, the kit is in powder form.

[0020] In a third aspect, the present application also provides a method for detecting Epstein-Barr virus. The method comprises extracting nucleic acid or plasmid from a test sample. Using the above-mentioned kit and the nucleic acid or plasmid from the test sample, an RPA amplification reaction is performed. After the reaction is complete, the fluorescent signal is analyzed.

[0021] In some embodiments, the RPA amplification reaction temperature is 30° C. to 40° C., and the reaction time is 5 min to 20 min.

[0022] In a fourth aspect, the embodiments of the present application also provide an application of a primer set and a probe in detecting Epstein-Barr virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A flow chart of the detection method provided in the embodiment of the present application;

[0024] Figure 2 A curve diagram of sensitivity test time and fluorescence value for detecting EB virus provided in an embodiment of the present application;

[0025] Figure 3 A curve diagram of the detection time of Epstein-Barr virus provided in an embodiment of the present application;

[0026] Figure 4 A curve chart of the detection time for detecting EB virus provided as a comparative example. DETAILED DESCRIPTION

[0027] The embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0028] 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 the technical features being referred to. Thus, a feature specified as "first" or "second" may explicitly or implicitly include one or more of such features. Throughout this application, unless otherwise specified, "plurality" means two or more.

[0029] In this application, DNA (deoxyribonucleic acid) is a long-chain polymer composed of four deoxynucleotides: adenine deoxynucleotide (dATP), thymine deoxynucleotide (dTTP), cytosine deoxynucleotide (dCTP), and guanine deoxynucleotide (dGTP).

[0030] A test kit is a box for containing chemical reagents for detecting chemical components, drug residues, virus types, etc. Of course, it will be understood by those skilled in the art that the box may also be other containers such as tubes for containing chemical reagents.

[0031] Epstein-Barr virus: (Epstein-Barr virus, EBV) belongs to the herpesvirus family, herpesvirus type IV, and is a herpesvirus that is tropic to human lymphocytes.

[0032] TaqMan probes: TaqMan fluorescent probes are oligonucleotide probes with a fluorophore at the 5' end, such as 5-carboxyfluorescein-5-succimidyl ester (5-FAM), tetrachloro-6-carboxyfluorescein (6-TET), VIC, or hexachloro-6-methylfluorescein (HEX). A quencher at the 3' end, such as 5-carboxytetramethylrhodamine (5-TAMRA) or BHQ carboxylic acid (BHQ), is carried out during PCR amplification. Fluorescence is released by cleavage of the 5' end of the probe, allowing detection of target mutations.

[0033] PCR: Polymerase Chain Reaction (PCR). PCR is a molecular biology technique used to amplify specific DNA fragments. It can be considered a specialized form of DNA replication outside of an organism. Its greatest feature is its ability to significantly increase the amount of DNA present in minute quantities.

[0034] RPA: Recombinase polymerase amplification (RPA).

[0035] Adenosine triphosphate: (Adenosine triphosphate, referred to as ATP).

[0036] Tetrahydrofuran: (tetrahydrofuran, referred to as THF).

[0037] FAM-dT: represents thymidine deoxynucleotide carrying the fluorescein group FAM (6-Carboxyfluorescein).

[0038] TET: Tetrachloro-6-carboxyfluorescein.

[0039] HEX: Hexachloro-6-methylfluorescein.

[0040] CY3: 3H-indocyanine.

[0041] JOE: 2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein.

[0042] BHQ-dT: represents a thymidine deoxynucleotide carrying a fluorescence quencher group BHQ (blackhole quencher).

[0043] C3-Spacer: indicates that the 3' terminal base is modified by a phosphate blocker.

[0044] Currently, the main laboratory diagnostic methods for EBV infection include EBV-specific antibody testing, heterophilic agglutination antibody testing, EBV nucleic acid load testing, and EBERs in situ hybridization testing. Recent research results indicate that serum / plasma EBV-DNA can be used for nasopharyngeal carcinoma screening, particularly for asymptomatic early-stage NPC patients, and may be more sensitive than the VCA-IgA assay. Furthermore, plasma EBV-DNA can be used for post-treatment monitoring and prognosis assessment of NPC patients. Furthermore, EBV-DNA nucleic acid load testing can serve as an important marker of tumor burden in patients with EBV-related lymphomas and can be used to assess treatment efficacy and predict prognosis.

[0045] Existing EBV-DNA detection is mainly carried out through real-time fluorescence PCR technology. That is, based on the working principle of Taqman probe, each round of amplification accumulates a fluorescent signal, which is used to monitor the entire PCR process in real time. Finally, the unknown template is qualitatively or quantitatively analyzed through the standard curve. Conventional PCR technology requires denaturation, annealing and extension steps, and its detection time is long, which cannot meet the urgent needs of EBV diagnosis.

[0046] In order to solve the problems existing in the prior art, the present application provides a primer set and a probe, wherein the primer set includes an amplification primer pair. The sequence of the amplification primer pair includes an upstream primer and a downstream primer.

[0047] Upstream primer: 5′-CCTCACATACACCTTACTGTTCACAACTCAGC-3′ SEQ ID No. 1.

[0048] Downstream primer: 5′-GTCCTAAGGAACAGCGATATCTCCCACCCCAT-3′ SEQ ID No. 2.

[0049] The probe sequences include:

[0050] 5'-GAAGAAGCAGGCGAAGATTCAGGAGAGTTCACTGCCCGCTCCTTGA-3'SEQ ID No. 3.

[0051] The primer set and probe provided in the embodiments of the present application can realize simultaneous amplification and detection without opening the lid, and can realize primer amplification and result detection in a fully closed state, overcoming the problem that the existing primer set needs to perform an amplification reaction first during detection, and then open the lid after the reaction results are obtained to transfer them to the detection platform for detection and analysis. Compared with existing detection, the detection time is shortened.

[0052] In some embodiments, the probe is labeled with a fluorescent group, a quencher, and THF. Both the fluorescent group and the quencher are labeled on a T base, with a 2-6 bp interval between the fluorescent group and the quencher. THF is located between the fluorescent group and the quencher. THF is ≥30 bp from the 5' end of the probe and ≥15 bp from the 3' end of the probe. THF is located between adjacent T bases. Fluorescent groups include FAM, TET, HEX, CY3, or JOE, and quenchers include BHQ1, BHQ2, TAMRA, DABCYL, MGB, or Eclipse. Selection is based on actual needs and is not specifically limited in this embodiment of the present application.

[0053] In some embodiments, the 3' end of the probe is further labeled with a blocking group, which blocks and seals the 3' end of the probe, preventing further extension of the 3' end of the probe.

[0054] In specific embodiments, the blocking group comprises an amine group, a phosphate group, biotin-TEG, a sulfhydryl group, or a C3-Spacer.

[0055] The present application also provides a kit comprising the above primer set and probe. The kit has a short detection time for EB virus and is easy to operate, and the details will not be described in detail.

[0056] In some embodiments, the kit is in powder form, which is convenient for use in detecting EB virus.

[0057] This application also provides a method for detecting EB virus. Figure 1 As shown, the detection method includes S1 to S3.

[0058] S1. Extracting nucleic acid or plasmid from the sample to be tested;

[0059] S2. Perform RPA amplification reaction using the kit and the nucleic acid or plasmid of the sample to be tested;

[0060] S3. After the reaction is completed, analyze the fluorescence signal.

[0061] The detection method provided in the present application uses a primer set and a probe to undergo RPA amplification reaction with the nucleic acid or plasmid of the sample to be tested, thereby analyzing the fluorescent signal. If a fluorescent signal is detected, the EB virus is detected; otherwise, the EB virus is not detected. The operation is simple.

[0062] There is no limitation on the RPA amplification reaction temperature in the above method, as long as it can achieve the RPA amplification reaction. The RPA amplification reaction temperature is 30°C to 40°C, and the reaction time is 5 minutes to 20 minutes. For example, the RPA amplification reaction temperature can be 30°C, 32°C, 36°C, or 40°C, and the reaction time can be 5 minutes, 10 minutes, 15 minutes, or 20 minutes. This application does not impose specific limitations on this.

[0063] The present application also provides an application of the above primer set and probe in detecting Epstein-Barr virus.

[0064] In order to make the implementation methods of the present application easier to understand, the present application will be described in detail below with reference to examples. These examples are only for illustration and are not intended to limit the scope of application of the present application.

[0065] Unless otherwise specified, the operations and processing methods involved in this application are conventional methods in the art.

[0066] Unless otherwise specified, the instruments used in this application are conventional instruments in this field.

[0067] Example 1

[0068] Based on the above, Example 1 of the present application provides a method for designing a primer set for detecting Epstein-Barr virus, including:

[0069] 1. Download the complete genome of gammaherpesvirus from the National Center for Biotechnology Information (NCBI) gene library. The genome length ranges from 170059 bp to 183865 bp. The specific genomes are shown in Table 1 below:

[0070] Table 1 Genome

[0071]

[0072]

[0073]

[0074] 2. The conserved regions were obtained using the multiple sequence alignment (ClustalW) algorithm. The conserved regions are as follows:

[0075] .

[0076] 3. Design primer sets within conserved regions

[0077] 3.1 Primer design includes the following steps:

[0078] (1) After sequence alignment of the amplicon region, the conserved region is selected;

[0079] (2) The upstream and downstream primers are 30–35 bases long, and the amplicon length is 80–400 bases;

[0080] (3) Avoid multiple Gs at the 5' end of the primer;

[0081] (4) The 3' end should contain G and C as much as possible;

[0082] (5) Avoid large amounts of palindromes and primer dimers;

[0083] (6) GC content is 40% to 60%.

[0084] 3.2 Probe design includes the following steps:

[0085] (1) The fluorescent group and the quenching group are labeled at the T base, 2 bp to 6 bp apart. The base between the fluorescent group and the quenching group is replaced by tetrahydrofuran (THF), and the replaced base is located between the two T bases.

[0086] (2) The probe should usually be 46 to 52 nucleotides long, with the 5' end at least 30 bp away from THF and the 3' end at least 15 bp away from THF;

[0087] (3) Avoid dimer formation between primers and probes;

[0088] (4) Blocking group at the 3' end of the fluorescent probe.

[0089] By designing the above-mentioned Epstein-Barr virus primer set, a primer set for detecting Epstein-Barr virus is obtained. For example, the fluorescent group is FAM, the quenching group is BHQ1, and the blocking group is C3-Spacer, which is not specifically limited in this application. The sequence of the amplification primer pair includes:

[0090] Upstream primer: 5′-CCTCACATACACCTTACTGTTCACAACTCAGC-3′;

[0091] Downstream primer: 5′-GTCCTAAGGAACAGCGATATCTCCCACCCCAT-3′.

[0092] The probe sequences include:

[0093] 5'-GAAGAAGCAGGCGAAGATTCAGGAGAGT[FAM-dT]C[THF]C[BHQ1-dT]GCCCGCTCCTTGA[C3 Spacer]-3'.

[0094] Based on the above, the primer set and probe provided in the embodiments of the present application bind to the amplification primer pair with the help of adenosine triphosphate and the localization factor (T4 UvsY) to form a recombinase primer complex, and search for homologous sequences in double-stranded DNA. Once the homologous sequence is located, the recombinase primer complex will insert into the double-stranded DNA to form a D-loop structure, initiating a chain displacement reaction, and the SSB protein will bind to the unwound DNA chain to prevent further displacement.

[0095] In addition, the recombinase is hydrolyzed from the recombinase-primer complex, exposing the 3' end primer and binding to the DNA polymerase. The DNA begins to replicate and extend, and eventually the two parent strands separate to form two new complementary double-stranded DNAs, achieving exponential amplification of the target region on the template. At the same time, the probe binds to the target sequence, and the base between the fluorescent group and the quencher group is replaced by tetrahydrofuran and remains bound. Tetrahydrofuran thus provides an enzyme cleavage site. When the probe binds to the target sequence, the enzyme cleavage site is cleaved, the fluorescent group separates from the quencher group, and a fluorescent signal is generated for detection. Therefore, positive detection can be completed in a short time. This shortens the detection time and simplifies the operation.

[0096] The following Examples 2 and 3 use the primer sets provided in the examples of this application to detect EB virus and perform sensitivity tests.

[0097] Example 2:

[0098] 1. Material preparation

[0099] The kit for detecting Epstein-Barr virus in this embodiment includes an RPA reaction solution, an upstream primer, a downstream primer, and a probe. The specific reaction components are shown in Table 2 below:

[0100] Table 2 Kit components and concentrations

[0101] Components concentration T4UvsX Recombinase 120 ng / μL T4UvsY Protein 60 ng / μL Single-chain binding protein (Gene32Protein) 600ng / μL BsuDNA Polymerase 0.25 U / μL dNTPs 200 μM Upstream primer 420nM Downstream primer 420nM probe 120nM Exonuclease III 1U / μL polyethylene glycol 5％ dithiothreitol 2mM Creatine phosphate 50mM Creatine kinase 100ng / μL

[0102] 2. Detection steps

[0103] (1) Prepare the components in Table 2 into an amplification system, package them into eight PCR tubes, and freeze-dry them in a freeze dryer under negative pressure to obtain lyophilized powder;

[0104] (2) Add 20 μL of the extracted nucleic acid or plasmid DNA directly to the lyophilized powder, mix gently and dissolve the lyophilized powder completely;

[0105] (3) Cover the 8-tube strip and centrifuge it. After centrifugation, place the 8-tube strip in a fluorescent PCR instrument and set the reaction temperature to 39°C and the reaction time to 20 min.

[0106] (4) The reaction is completed and the test results are analyzed. If there is a fluorescence curve, it is positive, which proves that EB virus is detected; if there is no fluorescence curve, it is negative, which proves that EB virus is not detected in the sample.

[0107] Example 3

[0108] The sensitivity test for detecting Epstein-Barr virus using the primer set provided in Example 1 of the present application includes the following steps:

[0109] (1) The conserved region sequence obtained in Example 1 was sent for plasmid synthesis. The plasmid was prepared at a rate of 3 × 10 5 copies / mL, 3×10 4 copies / mL, 3×10 3 copies / mL, 3×10 2 copies / mL for gradient dilution;

[0110] (2) Take 20 μL of the plasmid from step (1) and add it to the eight tubes containing lyophilized powder, mix gently and dissolve the lyophilized powder completely;

[0111] (3) Place the centrifuged eight-tube strip into a real-time fluorescence PCR instrument, set the reaction temperature to 39°C, and the reaction time to 20 min;

[0112] (4) Record the fluorescence data and analyze the results. Figure 2 shown.

[0113] from Figure 2 It can be seen that the sensitivity verification was carried out using plasmid DNA with four concentration gradients. The fluorescence increase of the amplification curve showed a decreasing trend as the concentration decreased, and basically showed an "S-shaped" amplification curve. When the concentration reached 3×10 2 copies / mL, it can still be detected normally, indicating that the primer set provided in Example 1 of the present application has good sensitivity for detecting EB virus.

[0114] Comparative Example

[0115] The test sample is 3×10 2 The detection method of Example 2 of the present application was used to compare the detection time with the conventional real-time fluorescence PCR detection method. The detection results of the detection method of Example 2 of the present application were as follows: Figure 3 shown.

[0116] Since the EB virus genome has different degrees of variation, the conserved region sequence refers to the DNA fragment that does not vary in different EB virus strains. This can ensure that the primer probe will not miss detection during amplification detection. Therefore, using the conserved region sequence in Example 1, the primer probe for fluorescent PCR was designed using Premier 5.0 software. The primer probe includes:

[0117] Upstream primer: 5′-CGTCCAAATTTTATTCTGGGG-3′, its concentration was 500 nM;

[0118] Downstream primer: 5′-CATTTTAGTCACAAGGGCAGTG-3′, its concentration was 500 nM;

[0119] Probe: 5'FAM-CACCTGAAACCTTGTTTTCGAGCACC-BHQ13', its concentration is 250 nM.

[0120] In addition, the amplification reagent was 2×RealUniversal PreMix, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0121] The conventional fluorescent PCR reaction steps are: 25℃ uracil carbonylase reaction for 3 minutes, 95℃ pre-denaturation for 2 minutes, 95℃ denaturation for 15 seconds, 60℃ annealing for 30 seconds, 60℃ extension for 30 seconds, and 45 cycles. The results are as follows: Figure 3 shown.

[0122] pass Figure 3 and Figure 4 It can be seen that the detection reaction time of Example 2 of the present application is 20 minutes, and the reaction time of the fluorescent PCR platform is 60 minutes. That is, the primer set provided in Example 1 of the present application is used to detect Epstein-Barr virus through RPA reaction, and its detection time is short, which has a time advantage in actual clinical application.

[0123] In the description of this specification, specific features, structures, materials or characteristics may be combined in an appropriate manner in any one or more embodiments or examples.

[0124] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims. Sequence Listing <110> Chengdu BOE Optoelectronics Technology Co., Ltd. BOE Technology Group Co., Ltd. <120> A primer set, probe and kit and their applications <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <400> 1 cctcacatac accttactgt tcacaactca gc 32 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <400> 2 gtcctaagga acagcgatat ctcccacccc at <210> 3 <211> 46 <212> DNA <213> Artificial Sequence <400> 3 gaagaagcag gcgaagattc aggagagttc actgcccgct ccttga

Claims

1. A primer set and a probe, characterized in that: The primer set includes an amplification primer pair; The sequences of the amplification primer pairs are as follows: Upstream primer: 5'-CCTCACATACACCTTACTGTTCACAACTCAGC-3'; and Downstream primer: 5'-GTCCTAAGGAACAGCGATATCTCCCACCCCAT-3'; The sequence of the probe is as follows: 5'-GAAGAAGCAGGCGAAGATTCAGGAGAGTTCACTGCCCGTCCCTT GA-3'.

2. The primer set and probe according to claim 1, characterized in that The probe is labeled with a fluorescent group, a quencher group and THF, wherein the fluorescent group and the quencher group are both labeled on T bases, the fluorescent group and the quencher group are separated by 2 bp to 6 bp, the THF is located between the fluorescent group and the quencher group, the THF is ≥30 bp away from the 5' end of the probe, the THF is ≥15 bp away from the 3' end of the probe, and the THF is located between adjacent T bases.

3. The primer set and probe according to claim 2, characterized in that: The fluorescent group is FAM, TET, HEX, CY3 or JOE, and the quenching group is BHQ1, BHQ2, TAMRA, DABCYL, MGB or Eclipse.

4. The primer set and probe according to claim 2 or 3, characterized in that: The 3' end of the probe is also labeled with a blocking group.

5. The primer set and probe according to claim 4, characterized in that: The blocking group is an amine group, a phosphate group, biotin-TEG, a sulfhydryl group or a C3-Spacer.

6. A kit, characterized in that include: The primer set and probe according to any one of claims 1 to 5.

7. The kit according to claim 6, characterized in that The kit is in powder form.

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