Primer group and kit for simultaneously detecting dengue virus and Zika virus and application of primer group and kit
By designing primer sets and using RNase H2 enzyme-mediated fluorescence detection technology, the problem of multiplex nucleic acid detection of dengue virus and Zika virus in resource-scarce areas has been solved, achieving rapid, simple, efficient and highly specific detection results.
Patent Information
- Application Number
- CN202511077666.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-01
- Publication Date
- 2025-11-07
AI Technical Summary
Current technologies cannot perform rapid and accurate multiplex nucleic acid testing in resource-scarce areas, especially for the on-site detection of dengue virus and Zika virus, which suffers from problems such as high equipment requirements, complex operation, low sensitivity, and high false positive rate.
Using primer set design, Splint R ligase catalyzes the ligation of target viral single-stranded RNA to specific DNA probes, forming a double-loop product. This product is then combined with RNase H2 enzyme and RNA-modified probes to achieve efficient amplification and highly specific fluorescence detection.
It enables rapid, simple, sensitive and highly specific multiplex nucleic acid detection under constant temperature conditions, reduces equipment requirements, improves detection efficiency and accuracy, and is suitable for on-site testing in resource-scarce areas.
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Figure CN120905447A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biotechnology, and particularly relates to a primer set for simultaneously detecting dengue virus and Zika virus, a kit and application thereof. BACKGROUND
[0002] Viral infectious diseases, especially mosquito-borne diseases such as dengue fever and Zika virus, pose a serious public health challenge worldwide, especially in relatively resource-poor tropical and subtropical regions. The outbreak of these diseases often comes suddenly and spreads rapidly in specific geographical areas.
[0003] Currently, for the detection of such viral RNA, reverse transcription real-time fluorescent PCR (RT-PCR) technology is considered the "gold standard". This technology first converts viral RNA into DNA through reverse transcriptase, then uses DNA polymerase for exponential amplification, and uses the 5'→3' exonuclease activity of the polymerase to hydrolyze a specific probe to generate a fluorescent signal. Although RT-PCR has high sensitivity and specificity, its application relies on expensive specialized instruments, precise control of cyclic temperature changes (rapid temperature changes), and a strict standard laboratory environment. These requirements make it difficult to widely deploy RT-PCR in resource-limited and poorly equipped areas, especially for on-site rapid diagnosis, and it cannot meet the needs of large-scale, instant screening. The delay in detection time can hinder early intervention and treatment, which is not conducive to disease control.
[0004] In addition to RT-PCR, viral isolation culture is a traditional diagnostic method, but this method can only detect viruses with infectious activity, has low sensitivity, and is complex to operate and has a long cycle (usually several days to more than a week), and requires a high level of laboratory environment and biosafety, which is also not suitable for rapid response. Immunological detection methods, such as antibody or antigen detection, although relatively simple to operate, have a "window period" problem, that is, in the early stage of infection, the host has not yet produced enough antibodies or the amount of viral antigen is insufficient to be detected, which can lead to false negative results, affecting the accuracy of early diagnosis.
[0005] In view of the above limitations of the prior art, especially the need for rapid and accurate diagnosis in resource-limited areas, the development of nucleic acid detection technology that can be performed under near-patient (Point-of-Care Testing, POCT) conditions, is simple to operate, results are fast and reliable, is of great significance for the effective monitoring, prevention and control of mosquito-borne infectious diseases. In recent years, the development of nucleic acid isothermal amplification technology, such as loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), and recombinase polymerase amplification (RPA), etc., due to its high efficiency of amplification at constant temperature, relatively low equipment requirements, and easy implementation of on-site detection, provides a new idea to solve the above problems. Among them, LAMP technology has shown great potential in rapid detection of pathogens due to its fast reaction speed, good specificity, and low requirements for operation and equipment, and is considered an ideal candidate for POCT platform.
[0006] However, in multiplex LAMP detection, primer design faces great challenges: LAMP itself has a large number of primers and long length, and the mutual interference between primers and primers and templates during multiplex amplification easily leads to amplification failure, increases the computational complexity and requires overall optimization design, and it is difficult to avoid primer dimer problems. In addition, Bst strand displacement DNA polymerase does not have the ability to hydrolyze probes, resulting in real-time LAMP detection often relying on colorimetric method or double-stranded DNA fluorescent dye, which has poor specificity and is prone to false positives. Although probe methods (such as introducing labeled strand displacement primers or designing molecular beacon probes) can provide signals, but will significantly affect the LAMP amplification efficiency, and the design of molecular beacon probes is complex, and the Tm value needs to be optimized to balance the stem loop formation and target binding. Although Cas enzyme system has attracted much attention in nucleic acid detection signal output, but it lacks selectivity in cutting the reporter probe, which brings technical obstacles to single-tube multiplex detection.
[0007] In summary, the prior art still cannot meet the needs of rapid, accurate, multiplex, and on-site nucleic acid detection of dengue, Zika virus and other mosquito-borne infectious diseases in specific scenarios (such as resource-poor areas). SUMMARY
[0008] The purpose of the present application is to provide a primer set for simultaneously detecting dengue virus and Zika virus, a kit and its application, which can solve the problems in the prior art. The kit using the primer set can perform rapid, accurate, multiplex, and on-site nucleic acid detection of dengue, Zika virus and other mosquito-borne infectious diseases in specific scenarios (such as resource-poor areas), and is convenient for popularization and application.
[0009] To achieve the above purpose, the technical scheme adopted by the present application is:
[0010] The first aspect of the present application provides a primer set for simultaneously detecting dengue virus and Zika virus, comprising primer pair 1 for dengue virus, primer pair 2 for Zika virus and a universal LAMP primer pair;
[0011] The nucleic acid sequence of primer pair 1 is:
[0012] The nucleotide sequence of DG-SLP-L is as shown in SEQ ID NO. 1;
[0013] The nucleotide sequence of DG-SLP-R is as shown in SEQ ID NO. 2;
[0014] The nucleic acid sequence of primer pair 2 is:
[0015] The nucleotide sequence of ZIKV-SLP-L is as shown in SEQ ID NO. 3;
[0016] The nucleotide sequence of ZIKV-SLP-R is as shown in SEQ ID NO. 4;
[0017] The nucleic acid sequence of the universal LAMP primer pair is:
[0018] The nucleotide sequence of FIP is as shown in SEQ ID NO. 5;
[0019] The nucleotide sequence of BIP is as shown in SEQ ID NO. 6.
[0020] The target virus single-stranded RNA is creatively used as a bridge molecule, under the catalysis of Splint R ligase, to guide and connect sequence-specific adjacent DNA probes, thereby directly forming a double-loop (dumbbell-shaped) structure product containing a universal circularization primer sequence.
[0021] The difficulty of primer dimer formation in traditional LAMP multiplex detection is completely avoided: by carefully designing universal LAMP primer sequences (FIP / BIP) at the ends of the connection probes, the connection products of multiple targets (such as DENV and ZIKV) are uniformly and efficiently amplified, which significantly improves the compatibility and efficiency of multiplex detection, and solves the core problem of mutual interference of multiplex LAMP primer design.
[0022] The amplification efficiency and speed are greatly improved: the connection step skillfully bypasses the initial rate-limiting step of generating double-loop primers by relying on displacement outer primers in traditional LAMP reactions, so that the entire amplification reaction can be quickly started at a constant temperature (such as 37℃). The universal primers FIP / BIP subsequently drive continuous polymerization and strand displacement reactions, efficiently producing a large number of stem-loop structure amplification products.
[0023] Real-time fluorescence detection with high specificity is realized: in order to overcome the defects that the Bst DNA polymerase cannot hydrolyze the probe and the traditional dye method is prone to false positives, the application innovatively combines the RNase H2 enzyme and the double-labeled DNA probe containing the RNA base (RNA-modified double-labeled probe). After the probe is fully complementary to the hybridization product, the RNase H2 can highly specifically cut the RNA base in the hybridization double-stranded chain, and the fluorescence signal is released. This RNase H2-mediated probe hydrolysis mechanism ensures that the detection signal is strictly corresponding to the target amplification product, and realizes the real-time, high-specificity fluorescence monitoring of multiple targets (such as DENV and ZIKV).
[0024] The second aspect of the application provides application of the primer set in preparation of a dengue virus and Zika virus detection kit.
[0025] The third aspect of the application provides a kit for simultaneously detecting dengue virus and Zika virus, comprising the primer set.
[0026] Further, the fluorescence probe DG-probe and the fluorescence probe ZIKV-probe are further included, the nucleotide sequence of the fluorescence probe DG-probe is obtained by replacing the deoxythymidine at the 8th position in the nucleotide sequence of SEQ ID NO. 7 with ribothymidine; the nucleotide sequence of the fluorescence probe ZIKV-probe is obtained by replacing the deoxythymidine at the 8th position in the nucleotide sequence of SEQ ID NO. 8 with ribothymidine.
[0027] Further, the Splint R ligase and the RNase H2 enzyme are further included.
[0028] The fourth aspect of the application provides application of the kit in detection of dengue and Zika viruses.
[0029] Further, the lower limit of the detection is 10 copies / reaction.
[0030] The fifth aspect of the application provides a simultaneous detection method of dengue virus and Zika virus based on ligase-mediated LAMP, comprising the following steps:
[0031] The components of the kit according to any one of claims 3-5 are mixed to configure a reaction system;
[0032] The amplification reaction is carried out under constant temperature conditions of 35-39 DEG C for 30-60 minutes;
[0033] Fluorescence detection is carried out.
[0034] Further, the amplification reaction is carried out under constant temperature conditions of 37 DEG C for 45 minutes.
[0035] Further, the reaction system further comprises a to-be-detected sample, and the to-be-detected sample is a serum sample.
[0036] In summary, by adopting the technical scheme, the application has the following beneficial effects:
[0037] The application provides a primer group, a kit and application thereof for simultaneously detecting dengue virus and Zika virus. BRIEF DESCRIPTION OF DRAWINGS
[0038] Figure 1 It is a schematic diagram of the ligase-mediated LAMP.
[0039] Figure 2 It is a process diagram of the RNase H2 and RNA modification probe for detecting the LAMP amplification product.
[0040] Figure 3 It is a sensitivity detection data diagram, wherein Figure 3 A in the figure is a real-time fluorescence signal data diagram of different concentrations of dengue virus, Figure 3 B in the figure is a fluorescence scanning curve data diagram of different concentrations of dengue virus, Figure 3 C in the figure is a linear relationship data diagram of different concentrations of dengue virus and fluorescence signal intensity, Figure 3 D in the figure is a real-time fluorescence signal data diagram of different concentrations of Zika virus, Figure 3 E in the figure is a fluorescence scanning curve data diagram of different concentrations of Zika virus, Figure 3 F in the figure is a linear relationship data diagram of different concentrations of Zika virus and fluorescence signal intensity.
[0041] Figure 4 It is a data diagram of the detection effect of the simulated clinical specimen. DETAILED DESCRIPTION
[0042] The application will be described in detail below with reference to the drawings.
[0043] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.
[0044] The first aspect of the embodiment provides a primer set for simultaneously detecting dengue virus and Zika virus, comprising primer pair 1 for dengue virus, primer pair 2 for Zika virus and universal LAMP primer pair;
[0045] The nucleic acid sequence of primer pair 1 is:
[0046] DG-SLP-L is the nucleotide sequence as shown in SEQ ID NO. 1;
[0047] DG-SLP-R is the nucleotide sequence as shown in SEQ ID NO. 2;
[0048] The nucleic acid sequence of primer pair 2 is:
[0049] ZIKV-SLP-L is the nucleotide sequence as shown in SEQ ID NO. 3;
[0050] ZIKV-SLP-R is the nucleotide sequence as shown in SEQ ID NO. 4;
[0051] The nucleic acid sequence of the universal LAMP primer pair is:
[0052] FIP is the nucleotide sequence as shown in SEQ ID NO. 5;
[0053] BIP is the nucleotide sequence as shown in SEQ ID NO. 6.
[0054] The second aspect of the embodiment provides the use of the above-mentioned primer set in the preparation of a dengue virus and Zika virus detection kit.
[0055] The third aspect of the embodiment provides a kit for simultaneously detecting dengue virus and Zika virus, comprising the above-mentioned primer set.
[0056] In some embodiments, the kit further comprises a fluorescent probe DG-probe and a fluorescent probe ZIKV-probe, wherein the nucleotide sequence of the fluorescent probe DG-probe is obtained by replacing the deoxythymidine at the 8th position of the nucleotide sequence of SEQ ID NO. 7 with ribothymidine; and the nucleotide sequence of the fluorescent probe ZIKV-probe is obtained by replacing the deoxythymidine at the 8th position of the nucleotide sequence of SEQ ID NO. 8 with ribothymidine. The fluorescent detection is achieved by RNase H2-mediated RNA modification probe technology, specifically: a double-labeled DNA probe containing RNA bases is designed to be complementary to the target gene sequence, and RNase H2 is used to specifically hydrolyze the RNA in the DNA-RNA double strand to release the fluorescent signal.
[0057] In some embodiments, the kit further comprises a Splint R ligase and an RNase H2 enzyme.
[0058] The fourth aspect of the present embodiment provides an application of the above-mentioned kit in detection of dengue and Zika viruses.
[0059] In some embodiments, the lower limit of detection is 10 copies / reaction.
[0060] The fifth aspect of the present embodiment provides a method for simultaneous detection of dengue and Zika viruses based on ligase-mediated LAMP, comprising the following steps:
[0061] Mixing the components of the kit according to any one of claims 3-5 to configure a reaction system;
[0062] Performing amplification reaction under constant temperature conditions of 35-39℃ for 30-60 minutes;
[0063] Performing fluorescent detection.
[0064] In some embodiments, the amplification reaction is performed under constant temperature conditions of 37℃ for 45 minutes.
[0065] In some embodiments, the reaction system further comprises a sample to be detected, and the sample to be detected is a serum sample.
[0066] The present application aims to meet the demand for rapid multiple detection of RNA of tropical mosquito-borne viruses, uses a Splint R ligase and a target single-stranded RNA as a bridge to efficiently connect two adjacent DNA probes to form a double-loop product. The universal LAMP primer sequence is designed at the end of the connecting probe to solve the difficulty of easy formation of dimers in multiple LAMP. The combination of RNase H2 and RNA-modified double-label probes realizes the dual fluorescent detection of dengue and Zika viruses.
[0067] In the presence of single-stranded DENV or Zika virus genomic RNA, the ligase facilitates the ligation of left and right DNA probes to form a double-looped product. This process bypasses the initial rate-limiting step of generating double-looped primers by displacing outer primers in traditional LAMP reactions. This innovative design enables the reaction to be performed at 37℃. Subsequently, continuous polymerization and strand displacement reactions between universal primers FIP and BIP generate a large amount of cauliflower-like LAMP amplification products, thereby achieving efficient amplification of the target gene Figure 1 To monitor the signal in real time, we used RNase H2-mediated signal output technology to detect the signal. The double-labeled DNA probe containing RNA bases can be fully complementary to the target gene sequence. By utilizing the unique property of RNase H2, which can specifically hydrolyze RNA in DNA-RNA double-stranded, but has no hydrolysis function on phosphodiester bonds in single-stranded or double-stranded DNA or RNA, we achieved real-time monitoring of the amplification process Figure 2
[0068] The purpose of the present application is to provide a simple and efficient isothermal LAMP dengue and Zika virus detection method. Compared with the currently commonly used fluorescent quantitative PCR method, the present application has the following characteristics and advantages:
[0069] (1) DNA ligase is used to catalyze the ligation of adjacent DNA probes to form a double-looped product in the presence of viral RNA, and universal primers are used to amplify dengue and Zika viruses.
[0070] (2) RNase H2-mediated RNA modification probe technology is used to detect the fluorescence signal.
[0071] Since the Bst strand displacement DNA polymerase used in LAMP technology does not have the ability to hydrolyze probes, real-time LAMP technology mainly relies on colorimetric and double-stranded DNA fluorescent dyes. These detection methods are not specific enough and can produce false positive results. The present application uses RNase H2-mediated RNA probe modification technology to realize signal output, ensuring the authenticity of the detection results.
[0072] In order to more clearly illustrate the above-mentioned embodiments, the following specific embodiment data is provided to better understand the scheme.
[0073] Example 1
[0074] This embodiment aims at the demand for rapid detection of multiple RNA of tropical mosquito-borne viruses. The splint R ligase and the target single-stranded RNA are used as a bridge to efficiently connect two adjacent DNA probes to form a double-loop product. The universal LAMP primer sequence is designed at the end of the connecting probe to solve the dilemma of easy dimer formation in multiple LAMP. The combination of RNase H2 and RNA-modified double-label probes realizes the dual fluorescence detection of dengue virus and Zika virus.
[0075] When single-stranded DENV or Zika virus genomic RNA exists, the ligase promotes the connection of left and right DNA probes to form a double-loop bottom product. This process bypasses the initial rate-limiting step of generating a double-loop primer by replacing the outer primer in the traditional LAMP reaction. This innovative design allows the reaction to be carried out at 37℃. Subsequently, the continuous polymerization and strand displacement reaction between the universal primers FIP and BIP produces a large amount of cauliflower-shaped LAMP amplification product, thereby achieving effective amplification of the target gene Figure 1 ). In order to monitor the signal in real time, the RNase H2-mediated signal output technology is used to detect the signal. The double-labeled DNA probe containing RNA bases can be fully complementary to the target gene sequence. By using the unique property of RNase H2, which can specifically hydrolyze RNA in DNA-RNA double-stranded, but has no hydrolysis function on the phosphodiester bond in single-stranded or double-stranded DNA or RNA, real-time monitoring of the amplification process is realized Figure 2 ).
[0076] The specific operation is as follows:
[0077] The reaction system for simultaneous detection of dengue and Zika viruses is shown in Table 1:
[0078] Table 1
[0079] Component Concentration / Amount 1x Isothermal Amplification Buffer 50 μL SLP-L Primer Pair 200 pM SLP-R Primer Pair 200 pM RNaseH2 0.5 mU / μL SplintR Ligase 25U Bsu DNA Polymerase Large Fragment 0.2 U / μL dNTPs 2 mM dUTPs 8 mM UNG 50 mU / μL ATP 1 mM FIP Primer 50 nM BIP Primer 50 nM Probe 100 nM
[0080] Reaction at 37℃ for 45min, and fluorescence detection.
[0081] The primer and probe sequences used are shown in Table 2:
[0082] Table 2
[0083]
[0084] The nucleotide sequence of the DG-probe is that the deoxythymidine (dT) at the 8th position in the nucleotide sequence of SEQ ID NO. 7 is replaced by ribothymidine (rT).
[0085] The nucleotide sequence of SEQ ID NO.7 is FAM-TGCACCAACACAGG-BHQ1. The nucleotide sequence of DG-probe is represented as FAM-TGCACCA / rT / ACACAGG-BHQ1.
[0086] The nucleotide sequence of ZIKV-probe is as follows: replace deoxythymidine (dT) at position 8 of the nucleotide sequence of SEQ ID NO.8 with ribothymidine (rT).
[0087] The nucleotide sequence of SEQ ID NO.8 is ROX-GAGGCGTAACTAAG-BHQ2. The nucleotide sequence of ZIKV-probe is represented as ROX-GAGGCGT / rA / AACTAAG-BHQ2.
[0088] Detection performance evaluation:
[0089] (1) Detection sensitivity:
[0090] To test the detection performance of the constructed method, dengue and Zika pseudoviruses were mixed into simulated serum, with the concentrations of each pseudovirus in the reaction system reaching 1E6 copies, 1E5 copies, 1E4 copies, 1E3 copies, 1E2 copies, and 1E1 copies, respectively. The detection results are as follows: Figure 3 As shown, with increasing dengue and Zika virus concentrations, the amplification curves start earlier and the signal intensity is higher. Furthermore, the fluorescence signal and viral load show a strong correlation, with the detection limit as low as 10 copies / reaction.
[0091] (2) Simulating the effect of clinical specimen testing:
[0092] Next, 24 simulated clinical samples, including those that were negative, dengue positive, Zika positive, and double positive, were tested. The test results are as follows: Figure 4 As shown, the detection accuracy for all clinical samples reached 100%, demonstrating excellent analytical performance.
[0093] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention 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; and these 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 the present invention.
Claims
1. A primer set for simultaneously detecting dengue virus and Zika virus, characterized by, The primer pair 1 is for dengue virus, the primer pair 2 is for Zika virus, and the universal LAMP primer pair; The nucleic acid sequence of the primer pair 1 is as follows: The nucleotide sequence of DG-SLP-L is as shown in SEQ ID NO. 1; The nucleotide sequence of DG-SLP-R is as shown in SEQ ID NO. 2; The nucleic acid sequence of the primer pair 2 is as follows: The nucleotide sequence of ZIKV-SLP-L is as shown in SEQ ID NO. 3; The nucleotide sequence of ZIKV-SLP-R is as shown in SEQ ID NO. 4; The nucleic acid sequence of the universal LAMP primer pair is as follows: The nucleotide sequence of FIP is as shown in SEQ ID NO. 5; The nucleotide sequence of BIP is as shown in SEQ ID NO.
6.
2. The primer set of claim 1 in the preparation of a dengue virus and Zika virus detection kit.
3. A kit for simultaneous detection of dengue virus and Zika virus, characterized in that, The primer set of claim 1 or 2.
4. The kit of claim 3, wherein Further comprising a fluorescent probe DG-probe and a fluorescent probe ZIKV-probe, the nucleotide sequence of the fluorescent probe DG-probe is obtained by replacing the deoxythymidine at the 8th position of the nucleotide sequence of SEQ ID NO. 7 with ribothymidine; the nucleotide sequence of the fluorescent probe ZIKV-probe is obtained by replacing the deoxythymidine at the 8th position of the nucleotide sequence of SEQ ID NO. 8 with ribothymidine.
5. The kit of claim 4, wherein Further comprising a Splint R ligase and an RNase H2 enzyme.
6. The kit of any one of claims 3-5 for use in the detection of dengue and Zika viruses.
7. Use according to claim 6, characterized in that, The lower limit of detection of the detection is 10 copies / reaction.
8. A method for simultaneous detection of dengue virus and Zika virus based on ligase-mediated LAMP, characterized by, The method comprises the following steps: Mixing the components of the kit of any one of claims 3-5 to configure a reaction system; Performing amplification reaction under constant temperature condition of 35-39℃ for 30-60 minutes; Performing fluorescence detection.
9. The method of claim 8, wherein, Performing amplification reaction under constant temperature condition of 37℃ for 45 minutes.
10. The method of claim 8, wherein, The reaction system further comprises a sample to be detected, and the sample to be detected is a serum sample.