A set of molecular targets for detecting hemorrhagic fever pathogens and their applications

Abstract

The present invention discloses a group of molecular targets for detecting hemorrhagic fever pathogens and their applications, wherein the hemorrhagic fever pathogens are Marburg virus, Rift Valley fever virus, yellow fever virus, West Nile virus, dengue virus and Zika virus. The present invention provides primers, primer-probe combinations and kits for detecting six hemorrhagic fever pathogens, and adds exogenous internal standards to monitor the nucleic acid extraction process and PCR amplification process to improve the accuracy of the results, optimizes the amplification system to speed up the detection speed, and the detection limit is as low as 400 copies / mL, with high sensitivity, good repeatability and strong specificity, providing a new technical option for the prevention and treatment of hemorrhagic fever.

Description

Technical Field

[0001] The present invention relates to the technical field of hemorrhagic fever virus diagnosis, and in particular to a group of molecular targets for detecting hemorrhagic fever pathogens and applications thereof. Background Art

[0002] Hemorrhagic fever (HF), also known as viral hemorrhagic fever (VHFs), is an acute infectious disease caused by a variety of viruses (such as dengue virus, etc.). Typical clinical manifestations include fever, bleeding, ecchymoses or petechiae on the skin and mucous membranes, and varying degrees of organ damage. Some viral hemorrhagic fevers are accompanied by varying degrees of hypotension and shock. There are many types of hemorrhagic fever pathogens, such as Marburg virus, Rift Valley fever virus, yellow fever virus, West Nile virus, dengue virus, Zika virus, etc., and the transmission methods are diverse. They are mainly transmitted by mosquitoes, sand flies and other organisms. Some viruses, such as Marburg virus, can also be transmitted through the patient's blood, secretions and other body fluids. After the virus enters the human body, it damages the blood vessel wall and coagulation function, causing bleeding in various organs. It enters the blood and activates cells that produce endogenous pyrogens, causing fever. Existing diagnostic methods generally use laboratory physiological and biochemical indicators and antigen-antibody testing to confirm the diagnosis. It is impossible to determine whether the patient is infected with hemorrhagic fever-related pathogens in the early stage of infection and the incubation period. Therefore, there is an urgent need for a method to sensitively and accurately detect hemorrhagic fever pathogens.

[0003] Real-time fluorescence PCR is a technology based on the principle of fluorescence resonance energy transfer (FRET). It is one of the commonly used methods for pathogen nucleic acid detection and can be used for laboratory examinations, epidemiological studies, exclusion diagnosis, etc. This method has high sensitivity and good specificity, and can accurately and quickly provide detection results of target viruses. Among them, the real-time fluorescence PCR technology based on TaqMan fluorescent labeled probes uses the thermostable DNA polymerase Taq enzyme to have polymerase activity in the 5'-3' direction while also having nuclease exonuclease activity in the 5'-3' direction for nucleotide sequences bound to the target sequence encountered during the polymerization extension process. The TaqMan fluorescent probe is labeled with a fluorescent emission group and a quenching group at the 5' and 3' ends, respectively, and the 3' end of the probe is phosphorylated to prevent the probe from being extended during PCR amplification. When the probe remains intact, the quenching group inhibits the fluorescence emission of the emission group. Once the emission group is separated from the quenching group, the inhibition is released, and the optical density at the emission wavelength of the fluorescent emission group increases and is detected by the fluorescence detection system. During the renaturation phase, the probe hybridizes with the template DNA, and during the extension phase, the Taq enzyme moves along the DNA template with the primer extension. When it moves to the probe position, the 5' exonuclease activity of the Taq DNA polymerase can degrade the specific fluorescent labeled probe, and the fluorescent group and the quenching group are separated, and the fluorescence is emitted. However, most of the real-time fluorescent PCR detection methods for hemorrhagic fever-related pathogens in the prior art can only detect a single or two pathogens, and cannot accurately detect multiple hemorrhagic fever pathogens at the same time. Summary of the invention

[0004] In order to solve the problem in the prior art that multiple hemorrhagic fever pathogens cannot be detected sensitively, accurately and simultaneously, the present invention provides a group of molecular targets for detecting hemorrhagic fever pathogens and applications thereof.

[0005] The first object of the present invention is to provide a set of molecular targets for detecting hemorrhagic fever pathogens.

[0006] The second object of the present invention is to provide the use of the reagent for detecting the molecular target in the preparation of a kit for detecting hemorrhagic fever pathogens.

[0007] The third object of the present invention is to provide a group of recombinant plasmids.

[0008] The fourth object of the present invention is to provide the use of the recombinant plasmid in preparing a kit for detecting hemorrhagic fever pathogens.

[0009] The fifth object of the present invention is to provide primers for detecting the molecular target.

[0010] The sixth object of the present invention is to provide the use of the primers in preparing products for detecting hemorrhagic fever pathogens.

[0011] The seventh object of the present invention is to provide a primer-probe combination for detecting the molecular target.

[0012] The eighth object of the present invention is to provide a kit for detecting hemorrhagic fever pathogens.

[0013] In order to achieve the above object, the present invention is implemented by the following scheme:

[0014] The invention designs detection primers and probes based on the full-length genome sequences, CDs, partial sequences and other similar gene sequences of the detection targets of different hemorrhagic fever pathogens in the NCBI library, selects conserved regions and specific segments through gene comparison, and performs specificity analysis, secondary structure analysis and dimer analysis.

[0015] The present invention requests protection for the following contents:

[0016] A group of molecular targets for detecting hemorrhagic fever pathogens, wherein the nucleotide sequences of the molecular targets are shown in SEQ ID NOs: 28 to 33, and the hemorrhagic fever pathogens are Marburg virus, Rift Valley fever virus, yellow fever virus, West Nile virus, dengue virus and Zika virus. Among them, the nucleotide sequence of the target sequence of Marburg virus is shown in SEQ ID NO: 28, the nucleotide sequence of the target sequence of Rift Valley fever virus is shown in SEQ ID NO: 29, the nucleotide sequence of the target sequence of yellow fever virus is shown in SEQ ID NO: 30, the nucleotide sequence of the target sequence of West Nile virus is shown in SEQ ID NO: 31, the nucleotide sequence of the target sequence of dengue virus is shown in SEQ ID NO: 32, and the nucleotide sequence of the target sequence of Zika virus is shown in SEQ ID NO: 33.

[0017] The reagent for detecting the molecular target is used in the preparation of a kit for detecting hemorrhagic fever plasmons, the nucleotide sequence of the target sequence is shown in SEQ ID NO: 28-33, and the hemorrhagic fever pathogens are Marburg virus, Rift Valley fever virus, yellow fever virus, West Nile virus, dengue virus and Zika virus.

[0018] A group of recombinant plasmids, wherein the recombinant plasmids are plasmids connected with molecular targets with nucleotide sequences as shown in SEQ ID NOs: 28-33.

[0019] Preferably, the recombinant plasmid uses pMD18-T plasmid as a backbone.

[0020] More preferably, the recombinant plasmid is based on the pMD18-T plasmid as the backbone, and any one of the molecular targets shown in the nucleotide sequence as SEQ ID NOs: 28 to 33 is set between the HindIII site and the BamHI site, namely, MBV-positive plasmid, RVFV-positive plasmid, YFV-positive plasmid, WNV-positive plasmid, Den-positive plasmid and ZIKV-positive plasmid.

[0021] The use of any of the recombinant plasmids in the preparation of a kit for detecting hemorrhagic fever pathogens, wherein the recombinant plasmid is used as a positive control and / or a positive standard. Among them, a plasmid connected to a molecular target with a nucleotide sequence such as SEQ ID NO: 28 is used as a positive control and / or a positive standard for Marburg virus; a plasmid connected to a molecular target with a nucleotide sequence such as SEQ ID NO: 29 is used as a positive control and / or a positive standard for Rift Valley fever virus; a plasmid connected to a molecular target with a nucleotide sequence such as SEQ ID NO: 30 is used as a positive control and / or a positive standard for yellow fever virus; a plasmid connected to a molecular target with a nucleotide sequence such as SEQ ID NO: 31 is used as a positive control and / or a positive standard for West Nile virus; a plasmid connected to a molecular target with a nucleotide sequence such as SEQ ID NO: 32 is used as a positive control and / or a positive standard for dengue virus; a plasmid connected to a molecular target with a nucleotide sequence such as SEQ ID NO: 33 is used as a positive control and / or a positive standard for Zika virus.

[0022] Preferably, the recombinant plasmid uses pMD18-T plasmid as a backbone.

[0023] More preferably, the recombinant plasmid is based on the pMD18-T plasmid as the backbone, and any one of the molecular targets shown in the nucleotide sequence as SEQ ID NOs: 28 to 33 is set between the HindIII site and the BamHI site, namely, MBV-positive plasmid, RVFV-positive plasmid, YFV-positive plasmid, WNV-positive plasmid, Den-positive plasmid and ZIKV-positive plasmid.

[0024] Primers for detecting the molecular targets whose nucleotide sequences are shown in SEQ ID NOs: 28-33.

[0025] Preferably, the nucleotide sequences of the primers are as shown in SEQ ID NOs: 1-2, SEQ ID NOs: 4-5, SEQ ID NOs: 7-8, SEQ ID NOs: 10-11, SEQ ID NOs: 13-14 and SEQ ID NOs: 16-17. Among them, the primers with nucleotide sequences as shown in SEQ ID NOs: 1-2 detect Marburg virus; the primers with nucleotide sequences as shown in SEQ ID NOs: 4-5 detect Rift Valley fever virus; the primers with nucleotide sequences as shown in SEQ ID NOs: 7-8 detect yellow fever virus; the primers with nucleotide sequences as shown in SEQ ID NOs: 10-11 detect West Nile virus; the primers with nucleotide sequences as shown in SEQ ID NOs: 13-14 detect dengue virus; the primers with nucleotide sequences as shown in SEQ ID NOs: 16-17 detect Zika virus.

[0026] The use of any of the primers in the preparation of products for detecting hemorrhagic fever pathogens should also be within the scope of protection of the present invention.

[0027] The use of any of the primers in the preparation of a kit for detecting hemorrhagic fever pathogens should also be within the scope of protection of the present invention.

[0028] The primer-probe composition for detecting the molecular target of the nucleotide sequence as shown in SEQ ID NO: 28 to 33 comprises a primer and a probe, wherein the primer is any of the above primers.

[0029] Preferably, the nucleotide sequences of the primers are shown in SEQ ID NOs: 1-2, SEQ ID NOs: 4-5, SEQ ID NOs: 7-8, SEQ ID NOs: 10-11, SEQ ID NOs: 13-14 and SEQ ID NOs: 16-17.

[0030] More preferably, the nucleotide sequence of the probe is as shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 18. Among them, the probe with the nucleotide sequence as shown in SEQ ID NO: 3 detects Marburg virus; the probe with the nucleotide sequence as shown in SEQ ID NO: 6 detects Rift Valley fever virus; the probe with the nucleotide sequence as shown in SEQ ID NO: 9 detects yellow fever virus; the probe with the nucleotide sequence as shown in SEQ ID NO: 12 detects West Nile virus; the probe with the nucleotide sequence as shown in SEQ ID NO: 15 detects dengue virus; the probe with the nucleotide sequence as shown in SEQ ID NO: 18 detects Zika virus.

[0031] Further preferably, the primer-probe composition comprises primer-probe groups 1 to 3;

[0032] The primer probe set 1 comprises primers with nucleotide sequences as shown in SEQ ID NOs: 1 to 2 and SEQ ID NOs: 16 to 17 and probes with nucleotide sequences as shown in SEQ ID NOs: 3 and SEQ ID NOs: 18;

[0033] The primer probe set 2 comprises primers with nucleotide sequences as shown in SEQ ID NOs: 4-5 and SEQ ID NOs: 13-14 and probes with nucleotide sequences as shown in SEQ ID NOs: 6 and SEQ ID NOs: 15;

[0034] The primer probe set 3 comprises primers with nucleotide sequences as shown in SEQ ID NOs: 7 to 8 and SEQ ID NOs: 10 to 11 and probes with nucleotide sequences as shown in SEQ ID NOs: 9 and SEQ ID NOs: 12;

[0035] The probes in each of the primer probe groups 1 to 3 are provided with different fluorescent groups at the 5' end and quenching groups at the 3' end; the fluorescent groups are TEXAS RED, CY5 or FAM; and the quenching groups are BHQ1 or BHQ2.

[0036] More preferably, the 5' end of the primer probe set 1 is provided with a fluorescent group FAM, and the 3' end is provided with a quencher group BHQ1; the 5' end of the primer probe set 2 is provided with a fluorescent group CY5, and the 3' end is provided with a quencher group BHQ2; the 5' end of the primer probe set 3 is provided with a fluorescent group TEXAS RED, and the 3' end is provided with a quencher group BHQ2.

[0037] The use of any of the primers or any of the primer-probe combinations in the preparation of a kit for detecting hemorrhagic fever should also be within the protection scope of the present invention.

[0038] A kit for detecting hemorrhagic fever pathogens, comprising reagents for detecting molecular targets of nucleotide sequences such as those shown in SEQ ID NOs: 28-33.

[0039] Preferably, the reagents comprise primers for detecting the molecular target.

[0040] More preferably, the nucleotide sequences of the primers are shown in SEQ ID NOs: 1-2, SEQ ID NOs: 4-5, SEQ ID NOs: 7-8, SEQ ID NOs: 10-11, SEQ ID NOs: 13-14 and SEQ ID NOs: 16-17.

[0041] More preferably, the reagent further comprises a probe for detecting the molecular target.

[0042] Further preferably, the nucleotide sequence of the probe is shown as SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15 and SEQ ID NO:18.

[0043] Preferably, the reagent comprises a primer-probe combination for detecting the molecular target.

[0044] More preferably, in the primer-probe composition, the nucleotide sequences of the primers are shown in SEQ ID NOs: 1-2, SEQ ID NOs: 4-5, SEQ ID NOs: 7-8, SEQ ID NOs: 10-11, SEQ ID NOs: 13-14 and SEQ ID NOs: 16-17.

[0045] Further preferably, in the primer-probe composition, the nucleotide sequence of the probe is shown in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15 and SEQ ID NO:18.

[0046] More preferably, the primer-probe composition comprises primer-probe groups 1 to 3;

[0047] The primer probe set 1 comprises primers with nucleotide sequences as shown in SEQ ID NOs: 1 to 2 and SEQ ID NOs: 16 to 17 and probes with nucleotide sequences as shown in SEQ ID NOs: 3 and SEQ ID NOs: 18;

[0048] The primer probe set 2 comprises primers with nucleotide sequences as shown in SEQ ID NOs: 4-5 and SEQ ID NOs: 13-14 and probes with nucleotide sequences as shown in SEQ ID NOs: 6 and SEQ ID NOs: 15;

[0049] The primer probe set 3 comprises primers with nucleotide sequences as shown in SEQ ID NOs: 7 to 8 and SEQ ID NOs: 10 to 11 and probes with nucleotide sequences as shown in SEQ ID NOs: 9 and SEQ ID NOs: 12;

[0050] The probes in each of the primer probe groups 1 to 3 are provided with different fluorescent groups at the 5' end and quenching groups at the 3' end; the fluorescent groups are TEXAS RED, CY5 or FAM; and the quenching groups are BHQ1 or BHQ2.

[0051] Still further preferably, the 5' end of the primer probe set 1 is provided with a fluorescent group FAM, and the 3' end is provided with a quencher group BHQ1; the 5' end of the primer probe set 2 is provided with a fluorescent group CY5, and the 3' end is provided with a quencher group BHQ2; the 5' end of the primer probe set 3 is provided with a fluorescent group TEXAS RED, and the 3' end is provided with a quencher group BHQ2.

[0052] Still further preferably, the working concentration of the primers of the primer probe sets 1 to 3 is 0.08 μM to 0.12 μM; the working concentration of the probes of the primer probe sets 1 to 3 is 0.08 μM to 0.12 μM.

[0053] Most preferably, the working concentration of the primers in the primer probe sets 1 to 3 is 0.1 μM each; the working concentration of the probes in the primer probe sets 1 to 3 is 0.1 μM each.

[0054] Preferably, the kit further comprises a recombinant plasmid connected to a molecular target with a nucleotide sequence such as SEQ ID NO: 28 to 33, and the recombinant plasmid serves as a positive control and / or a positive standard.

[0055] More preferably, the recombinant plasmid uses pMD18-T plasmid as a backbone.

[0056] Further preferably, the recombinant plasmid uses pMD18-T plasmid as a backbone, and any one of the molecular targets having a nucleotide sequence as shown in SEQ ID NOs: 28 to 33 is disposed between the HindIII site and the BamHI site.

[0057] Preferably, the kit further comprises an exogenous internal standard, which is a recombinant plasmid connected with an exogenous internal standard gene. The present invention does not specifically limit the exogenous internal standard gene, such as papain gene, Hordeumvulgare dehydrin gene (i.e., Dhn gene) and other conventional exogenous internal standard genes, which can achieve the purpose of detecting hemorrhagic fever in the present invention.

[0058] More preferably, the exogenous internal standard gene is the Dhn gene, NCBI No.: AF043095.1.

[0059] Still more preferably, the recombinant plasmid uses pMD18-T plasmid as a backbone.

[0060] Most preferably, the recombinant plasmid uses pMD18-T plasmid as a backbone, and the sequence of the Dhn gene (SEQ ID NO: 36) is located between the HindIII site and the BamHI site, ie, the Dhn plasmid.

[0061] More preferably, the primer-probe combination further comprises a primer combination 4 for detecting the Dhn gene, comprising primers having nucleotide sequences as shown in SEQ ID NOs: 25 to 26 and a probe having a nucleotide sequence as shown in SEQ ID NO: 27.

[0062] Still more preferably, the 5'-end fluorescent group of the probe with the nucleotide sequence as shown in SEQ ID NO: 27 is VIC, and the 3' quenching group is BHQ1.

[0063] Still more preferably, in primer probe set 4, the working concentration of the primers with nucleotide sequences as shown in SEQ ID NOs: 25-26 is 0.08 μM to 0.12 μM; the working concentration of the probe with nucleotide sequences as shown in SEQ ID NO: 27 is 0.08 μM to 0.12 μM.

[0064] Most preferably, in primer probe set 4, the working concentration of the primers with nucleotide sequences as shown in SEQ ID NOs: 25 to 26 is 0.1 μM each; the working concentration of the probe with nucleotide sequence as shown in SEQ ID NO: 27 is 0.1 μM.

[0065] Preferably, the kit further comprises PCR Buffer and PCR enzyme.

[0066] More preferably, the PCR Buffer comprises Tris-HCl, Tween 20, KCl, (NH4)2SO4 and MgCl2.

[0067] Further preferably, the PCR Buffer further comprises DEPC H2O.

[0068] More preferably, the PCR reaction enzyme comprises hot start Taq enzyme, Taq enzyme, c-MMLV enzyme, dNTPs and Rnasin.

[0069] More preferably, the PCR reaction enzyme further comprises DEPC water.

[0070] More preferably, the method for using the kit comprises the following steps:

[0071] S1. Obtaining nucleic acid of the sample to be tested;

[0072] S2. Using the nucleic acid of the sample to be tested as a template, the above-mentioned primer-probe combination, PCR Buffer and PCR enzyme are used to prepare a reaction system for fluorescent quantitative PCR amplification.

[0073] The present invention has no particular limitation on the method for extracting nucleic acid. Generally, the nucleic acid of the sample to be tested can be extracted using conventional laboratory methods or a nucleic acid extraction kit.

[0074] Still further preferably, in step S2, the program of fluorescence quantitative PCR amplification is: 48-52°C, 2 minutes; 93-97°C, 2 minutes; 93-97°C, 5 seconds, 58-62°C, 10-35 seconds, 45 cycles.

[0075] Most preferably, in step S2, the program of the fluorescent quantitative PCR amplification is: 50°C, 2 minutes; 95°C, 2 minutes; 95°C, 5 seconds, 60°C, 10 seconds, 45 cycles, and collecting fluorescence.

[0076] Compared with the prior art, the present invention has the following beneficial effects:

[0077] The present invention provides primers, primer-probe combinations and kits for detecting six hemorrhagic fever pathogens, and adds exogenous internal standards to monitor the nucleic acid extraction process and the PCR amplification process to improve the accuracy of the results, optimizes the amplification system to speed up the detection speed, and has a detection limit as low as 400 copies / mL, high sensitivity, good repeatability and strong specificity, providing a new technical option for the detection of hemorrhagic fever pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 This is a map of the pMD18-T plasmid.

[0079] Figure 2 These are the detection results of reaction system 1 and reaction system 2 before pathogen optimization; A is the mixed channel amplification curve of reaction system 1 detecting MBV, Den and YHCHIKV, with Dhn as the internal standard; B to D are the single channel amplification curves of YHCHIKV, Den and MBV, respectively; E is the mixed channel amplification curve of reaction system 2 detecting WNV, RVFV and S, with Dhn as the internal standard; F to H are the single channel amplification curves of S, WNV and RVFV, respectively.

[0080] Figure 3 Figure 1 is the detection results of reaction system 1 and reaction system 2 after pathogen optimization; A is the mixed channel amplification curve of reaction system 1 detecting MBV, Den and YFV, with Dhn as the internal standard, B to D are the single channel amplification curves of YFV, Den and MBV respectively; E is the mixed channel amplification curve of reaction system 2 detecting WNV, RVFV and ZIKV, with Dhn as the internal standard, F to H are the single channel amplification curves of ZIKV, WNV and RVFV respectively.

[0081] Figure 4 Figure 2 is the optimization status of the multiplex real-time fluorescence quantitative PCR reaction system; A is the detection result of reaction system 1 before optimization; B is the detection result of reaction system 1 after optimization; C is the detection result of reaction system 2 before optimization; D is the detection result of reaction system 2 after optimization.

[0082] Figure 5 These are the sensitivity evaluation results for detecting hemorrhagic fever pathogens; A is the result of detecting gradient dilution plasmids in reaction system 1, the detected pathogens include MBV, Den and YFV, and the internal standard is Dhn; B is the result of detecting gradient dilution plasmids in reaction system 2, the detected pathogens include ZIKV, RVFV and WNV, and the internal standard is Dhn.

[0083] Figure 6 The results of repeatability evaluation for detecting hemorrhagic fever pathogens; A is the result of 10 repeated tests in reaction system 1, in which the detected pathogens include MBV, Den and YFV, and the internal standard is Dhn; B is the result of 10 repeated tests in reaction system 2, in which the detected pathogens include ZIKV, RVFV and WNV, and the internal standard is Dhn.

[0084] Figure 7 Specificity evaluation results for detecting hemorrhagic fever pathogens: detection results of pathogens with similar clinical manifestations using reaction system 1 and reaction system 2, with Dhn as the internal standard. DETAILED DESCRIPTION

[0085] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0086] Example 1 Determination of primers, probes and reaction systems for detecting hemorrhagic fever pathogens

[0087] 1. Design of primers and probes

[0088] Marburg virus (MBV), Rift Valley fever virus (RVFV), yellow fever virus (YFV), West Nile virus (WNV), dengue virus (Den), Zika virus (ZIKV), chikungunya virus (YHCHIKV) and Hantan virus (S) were used as candidate pathogens for detecting hemorrhagic fever, and detection primers and probes were designed according to the conserved regions of each virus as shown in Table 1. The artificially synthesized Dhn plasmid was used as an exogenous internal standard, and the Dhn plasmid was composed of a fragment sequence (SEQ ID NO: 36) of the Dhn gene (NCBI No.: AF043095.1) inserted into the Figure 1 The pMD18-T plasmid shown was obtained between the HindIII site and the BamHI site.

[0089] Table 1 Sequence information of primers and probes for detecting hemorrhagic fever pathogens

[0090]

[0091] The probes in Table 1 are all Taqman probes, wherein the 5'-end fluorescent group of the probe of MBV (SEQ ID NO: 3), the probe of ZIKV (SEQ ID NO: 18) and the probe of S (SEQ ID NO: 24) is FAM, and the 3'-end quenching group is BHQ1; the 5'-end fluorescent group of the probe of RVFV (SEQ ID NO: 6) and the probe of Den (SEQ ID NO: 15) is CY5, and the 3'-end quenching group is BHQ2; the 5'-end fluorescent group of the probe of YFV (SEQ ID NO: 9), the probe of WNV (SEQ ID NO: 12) and the probe of YHCHIKV (SEQ ID NO: 21) is TEXAS RED, and the 3'-end quenching group is BHQ2; the 5'-end fluorescent group of the probe of Dhn (SEQ ID NO: 27) is VIC, and the 3'-end quenching group is BHQ1.

[0092] 2. Determination of target pathogens

[0093] 1. Experimental methods

[0094] The target sequences of MBV, RVFV, YFV, WNV, Den, ZIKV, YHCHIKV and S (SEQ ID NOs: 28-35) were inserted into Figure 1 Between the HindIII site and the BamHI site of the pMD18-T plasmid shown, the positive plasmids of each pathogen are obtained, which are recorded as MBV positive plasmid, RVFV positive plasmid, YFV positive plasmid, WNV positive plasmid, Den positive plasmid, ZIKV positive plasmid, YHCHIKV positive plasmid and S positive plasmid in sequence.

[0095] The positive plasmids of each pathogen and the Dhn plasmid were used as templates to perform real-time fluorescence quantitative PCR amplification using the primers and probes in Table 1. The real-time fluorescence quantitative PCR reaction program is shown in Table 2, and the PCR reaction system is shown in Table 3.

[0096] Table 2 Real-time fluorescence quantitative PCR reaction procedure

[0097]

[0098] Table 3 Real-time fluorescence quantitative PCR reaction system

[0099]

[0100]

[0101] 2. Experimental results

[0102] like Figure 2As shown in A to D in the figure, in the reaction system 1 mixed with multiple primer probes, the pathogens Marburg virus (MBV), dengue virus (Den) and chikungunya virus (YHCHIKV) and the exogenous internal standard Dhn all have amplification curves, but the Ct value of chikungunya virus (YHCHIKV) is relatively low overall, and at low and medium concentrations (less than 4×10 3 When the template dosage was 200 copies / mL, the amplification curve did not show obvious concentration dependence. Figure 2 As shown in Figures E to H, in the reaction system 2 mixed with multiple primer probes, the pathogens West Nile virus (WNV), Rift Valley fever virus (RVFV) and Hantaan virus (S) and the exogenous internal standard Dhn all have amplification curves, but the Ct value of Hantaan virus (S) is relatively late overall, and at low and medium concentrations (less than 4×10 3 The amplification curve did not show obvious concentration dependence at a template dosage of 100 copies / mL. Based on the above results, it was decided to eliminate the chikungunya virus and renal syndrome Hantan virus, and not use the fragment sequence (SEQ ID NO: 34) of the unassigned DNA gene (NCBI No.: OF451348.1) and the fragment sequence (SEQ ID NO: 35) of the S gene (NCBI No.: JQ083395.1) as molecular targets for detecting hemorrhagic fever pathogens.

[0103] like Figure 3 As shown in A to D in Figure 1, in reaction system 1, after yellow fever virus (YFV) was used instead of chikungunya virus (YHCHIKV), the three pathogens and the exogenous internal standard Dhn all had amplification curves, and the Ct values ​​were basically the same, showing obvious concentration dependence as a whole. Figure 3 As shown in Figures E to H, in reaction system 2, after Zika virus (ZIKV) was used instead of Hantaan virus (S), the three pathogens and the exogenous internal standard Dhn all had amplification curves, and the Ct values ​​were basically the same, showing obvious concentration dependence as a whole.

[0104] In summary, Marburg virus, Rift Valley fever virus, yellow fever virus, West Nile virus, dengue virus and Zika virus all amplified smooth and effective curves, so these six viruses were used as target pathogens for detecting hemorrhagic fever in subsequent experiments, and the gene target sequences corresponding to each virus (as shown in Table 1) were used as molecular targets for detecting hemorrhagic fever pathogens.

[0105] 3. Optimization of multiplex real-time fluorescence quantitative PCR reaction system

[0106] 1. Experimental methods

[0107] The present invention does not specifically limit the combination of primers and probes for detecting six target pathogens (Marburg virus, Rift Valley fever virus, yellow fever virus, West Nile virus, dengue virus and Zika virus). This embodiment provides one of the combinations, which is as follows:

[0108] Reaction system 1: primers and probes of SEQ ID NOs: 1 to 3, SEQ ID NOs: 7 to 9 and SEQ ID NOs: 13 to 15, for simultaneous detection of Marburg virus, yellow fever virus and dengue virus;

[0109] Reaction system 2: primers and probes of SEQ ID NOs: 4-6, SEQ ID NOs: 10-12, and SEQ ID NOs: 16-18, for simultaneous detection of Rift Valley fever virus, West Nile virus, and Zika virus;

[0110] Reaction system 1 and reaction system 2 were independently used for PCR amplification.

[0111] Based on the combination of the above primers and probes, the concentrations of primers and probes in the multiplex real-time fluorescence quantitative PCR reaction system were optimized. The reaction system before optimization is shown in Table 4, and the reaction system after optimization is shown in Table 5. Amplification was performed on an ABI7500 fluorescence PCR instrument, and the PCR reaction program was shown in Table 2.

[0112] Table 4 Multiplex real-time fluorescence quantitative PCR reaction system before optimization

[0113]

[0114]

[0115] Table 5 Optimized multiplex real-time fluorescence quantitative PCR reaction system

[0116]

[0117]

[0118] 2. Experimental results

[0119] like Figure 4 As shown in A and B in the figure, compared with the reaction system before optimization, the optimized reaction system 1 has a smooth S-shaped curve, a plateau phase, an obvious amplification gradient, no obvious Ct difference between different detection target channels, and basically the same fluorescence height; Figure 4 As shown in C and D in Figure 5, compared with the reaction system before optimization, the optimized reaction system 2 has a smooth S-shaped curve, a plateau phase, an obvious amplification gradient, no obvious Ct difference between different detection target channels, and basically the same fluorescence height. Therefore, the reaction system shown in Table 5 was used for sensitivity evaluation, repeatability evaluation, and specificity evaluation.

[0120] Example 2 Evaluation of sensitivity of detecting hemorrhagic fever pathogens

[0121] 1. Experimental Methods

[0122] The MBV-positive plasmid, RVFV-positive plasmid, YFV-positive plasmid, WNV-positive plasmid, Den-positive plasmid and ZIKV-positive plasmid prepared in Example 1 were diluted to appropriate concentrations and then diluted 10-fold to obtain gradient dilution plasmids of 6 pathogens, with concentrations of 4×10 5 copies / mL, 4×10 4 copies / mL, 4×10 3 copies / mL and 400copies / mL.

[0123] Using the gradient dilution plasmids of the six pathogens obtained as templates, the reaction system was prepared according to the information shown in Table 5, and the detection was performed on an ABI7500 fluorescent PCR instrument according to the reaction program shown in Table 2.

[0124] 2. Experimental Results

[0125] like Figure 5 As shown in A, the curve of reaction system 1 in the test results of 400 copies / mL sample concentration is S-shaped, with an obvious plateau period, no Ct difference between different channels, and good repeatability; Figure 5 As shown in B, the curve of reaction system 2 in the detection result of the sample with a concentration of 400 copies / mL is S-shaped, with an obvious plateau period, no Ct difference between different channels, and good repeatability. The above results show that the method for detecting hemorrhagic fever pathogens of the present invention has high sensitivity, and the detection limit is as low as 400 copies / mL, which meets the detection requirements.

[0126] Example 3 Evaluation of Repeatability of Detection of Hemorrhagic Fever Pathogens

[0127] 1. Experimental Methods

[0128] The MBV positive plasmid, RVFV positive plasmid, YFV positive plasmid, WNV positive plasmid, Den positive plasmid and ZIKV positive plasmid prepared in Example 1 were adjusted to a concentration of ×10 2 copies / mL, and then used as a template to prepare the reaction system according to the information shown in Table 5, and tested on an ABI7500 fluorescent PCR instrument according to the reaction program shown in Table 2, and repeated the test 10 times.

[0129] 2. Experimental Results

[0130] like Figure 6As shown in A, the reaction system 1 was repeated to detect the sample 10 times, and the CV values ​​of different fluorescence channels were counted respectively, and the coefficient of variation of the CV value was <5%; Figure 6 As shown in B, the reaction system 2 repeatedly tested the sample 10 times, and the CV values ​​of different fluorescence channels were counted respectively, and the coefficient of variation of the CV value was <5%. The above results show that the method for detecting hemorrhagic fever pathogens of the present invention has good repeatability and meets the detection requirements.

[0131] Example 4 Evaluation of specificity for detecting hemorrhagic fever pathogens

[0132] 1. Experimental Methods

[0133] The clinical manifestations of Ebola virus, Lassa fever virus, Hantan virus with renal syndrome, Chikungunya virus, Marburg virus, Rift Valley fever virus, yellow fever virus, West Nile virus, dengue virus and Zika virus are similar, and the infected people all have hemorrhagic fever symptoms.

[0134] The sequence of the Ebola virus Vp35 gene (NCBI No.: MN416402.1) (SEQ ID NO: 37) and the gene sequence of the Lassa fever virus L protein (NCBI No.: NC_004297.1) (SEQ ID NO: 38) were inserted into Figure 1 Between the HindIII site and the BamHI site of the pMD18-T plasmid shown, an EBoV-positive plasmid and a LASV-positive plasmid are obtained.

[0135] The EBoV positive plasmid and LASV positive plasmid as well as the YHCHIKV positive plasmid and S positive plasmid prepared in Example 1 were adjusted to a concentration of 10 6 copies / mL, and then used as a template to prepare a reaction system according to the information shown in Table 5, and detected on an ABI7500 fluorescent PCR instrument according to the reaction program shown in Table 2.

[0136] 2. Experimental Results

[0137] like Figure 7 As shown, the detection results of reaction system 1 and reaction system 2 for pathogens with similar clinical manifestations (Ebola virus, Lassa fever virus, Hantan virus with renal syndrome and Chikungunya virus) were negative, and there was no amplification curve. The above results show that the method of the present invention for detecting hemorrhagic fever pathogens has high specificity and meets the detection requirements.

[0138] Example 5 A kit for detecting hemorrhagic fever pathogens

[0139] 1. Composition

[0140] (1) Primers and probes

[0141] Primers and probes for detecting Marburg virus (SEQ ID NOs: 1-3), primers and probes for detecting Rift Valley fever virus (SEQ ID NOs: 4-6), primers and probes for detecting yellow fever virus (SEQ ID NOs: 7-9), primers and probes for detecting West Nile virus (SEQ ID NOs: 10-12), primers and probes for detecting dengue virus (SEQ ID NOs: 13-15), primers and probes for detecting Zika virus (SEQ ID NOs: 16-18), and primers and probes for detecting exogenous internal standard Dhn10 (SEQ ID NOs: 25-27).

[0142] (2)PCR Buffer

[0143] The main components are Tris-HCl (1000 μM), Tween 20 (0.5%), KCl (500 μM), (NH4)2SO4 (500 μM), MgCl2 (500 μM) and DEPC H2O.

[0144] (3) PCR enzyme

[0145] The main components are hot-start Taq enzyme, Taq enzyme, c-MMLV enzyme, dNTPs, Rnasin and DEPC water.

[0146] (4) Exogenous internal standard

[0147] The Dhn plasmid of Example 1.

[0148] (5) Positive control

[0149] The MBV-positive plasmid, RVFV-positive plasmid, YFV-positive plasmid, WNV-positive plasmid, Den-positive plasmid and ZIKV-positive plasmid of Example 1.

[0150] 2. Usage

[0151] 1. Extraction of Nucleic Acids

[0152] This kit does not specify the method for extracting nucleic acids. Generally, conventional laboratory methods or nucleic acid extraction kits can be used to extract nucleic acids from the samples to be tested.

[0153] 2. Preparation of reaction system

[0154] The reaction system was prepared according to the information shown in Table 5.

[0155] 3. Detection

[0156] On the fluorescence PCR instrument, set the corresponding fluorescence detection channel according to the fluorescent group labeled on the probe, and perform detection according to the reaction program shown in Table 2.

[0157] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A primer-probe combination for detecting a molecular target of a hemorrhagic fever pathogen, characterized in that: The hemorrhagic fever pathogens are Marburg virus, Rift Valley fever virus, yellow fever virus, West Nile virus, dengue virus and Zika virus; the nucleotide sequences of the molecular targets are shown in SEQ ID NOs: 28 to 33, respectively; The primer-probe composition comprises primers and probes, wherein the nucleotide sequences of the primers are as shown in SEQ ID NOs: 1-2, SEQ ID NOs: 4-5, SEQ ID NOs: 7-8, SEQ ID NOs: 10-11, SEQ ID NOs: 13-14 and SEQ ID NOs: 16-17; wherein the primers with nucleotide sequences as shown in SEQ ID NOs: 1-2 detect Marburg virus; the primers with nucleotide sequences as shown in SEQ ID NOs: 4-5 detect Rift Valley fever virus; the primers with nucleotide sequences as shown in SEQ ID NOs: 7-8 detect yellow fever virus; the primers with nucleotide sequences as shown in SEQ ID NOs: 10-11 detect West Nile virus; the primers with nucleotide sequences as shown in SEQ ID NOs: 13-14 detect dengue virus; the primers with nucleotide sequences as shown in SEQ ID NOs: 16-17 detect Zika virus; The primers are fluorescent quantitative PCR primers; The nucleotide sequences of the probes are as shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 18, wherein the probe with the nucleotide sequence as shown in SEQ ID NO: 3 detects Marburg virus; the probe with the nucleotide sequence as shown in SEQ ID NO: 6 detects Rift Valley fever virus; the probe with the nucleotide sequence as shown in SEQ ID NO: 9 detects yellow fever virus; the probe with the nucleotide sequence as shown in SEQ ID NO: 12 detects West Nile virus; the probe with the nucleotide sequence as shown in SEQ ID NO: 15 detects dengue virus; and the probe with the nucleotide sequence as shown in SEQ ID NO: 18 detects Zika virus.

2. The primer-probe combination according to claim 1, characterized in that The primer-probe combination comprises primer-probe groups 1 to 3; The primer probe set 1 comprises primers with nucleotide sequences as shown in SEQ ID NOs: 1 to 2 and SEQ ID NOs: 16 to 17 and probes with nucleotide sequences as shown in SEQ ID NOs: 3 and SEQ ID NOs: 18; The primer probe set 2 comprises primers with nucleotide sequences as shown in SEQ ID NOs: 4-5 and SEQ ID NOs: 13-14 and probes with nucleotide sequences as shown in SEQ ID NOs: 6 and SEQ ID NOs: 15; The primer probe set 3 comprises primers with nucleotide sequences as shown in SEQ ID NOs: 7 to 8 and SEQ ID NOs: 10 to 11 and probes with nucleotide sequences as shown in SEQ ID NOs: 9 and SEQ ID NOs: 12; The probes in each of the primer probe groups 1 to 3 are provided with different fluorescent groups at the 5' end and quenching groups at the 3' end; the fluorescent groups are TEXAS RED, CY5 or FAM; and the quenching groups are BHQ1 or BHQ2.

3. A kit for detecting hemorrhagic fever pathogens, characterized in that: Comprising the primer-probe composition according to claim 1; The detection of the kit is divided into reaction system 1 and reaction system 2; the reaction system 1 includes primers and probes with nucleotide sequences such as SEQ ID NOs: 1 to 3, SEQ ID NOs: 7 to 9 and SEQ ID NOs: 13 to 15; the reaction system 2 includes primers and probes with nucleotide sequences such as SEQ ID NOs: 4 to 6, SEQ ID NOs: 10 to 12 and SEQ ID NOs: 16 to 18.

4. The kit according to claim 3, characterized in that It also comprises a recombinant plasmid connected with a molecular target with a nucleotide sequence such as SEQ ID NO: 28 to 33, wherein the recombinant plasmid is used as a positive control or a positive standard.

5. The kit according to claim 3, characterized in that It also comprises an exogenous internal standard, which is a recombinant plasmid connected with an exogenous internal standard gene; the exogenous internal standard gene is a Dhn gene, NCBI No.: AF043095.

1.

6. The kit according to claim 5, characterized in that It also comprises primers and probes for detecting the Dhn gene. The nucleotide sequences of the primers for detecting the Dhn gene are shown in SEQ ID NOs: 25-26, and the nucleotide sequence of the probe for detecting the Dhn gene is shown in SEQ ID NO: 27.

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