Primer group and kit for nucleic acid detection of severe fever with thrombocytopenia syndrome virus

Abstract

The invention provides a primer group and a kit for nucleic acid detection of severe fever with thrombocytopenia syndrome virus, and belongs to the technical field of nucleic acid visual detection. The invention provides a primer group for nucleic acid detection of a severe fever with thrombocytopenia syndrome virus and a nucleic acid detection kit of the severe fever with thrombocytopenia syndrome virus. The problems that in the prior art, when the severe fever with thrombocytopenia syndrome virus is detected, operation is complex, sensitivity is low, and a false positive result is caused by formed aerosol are solved. According to the detection method established by the invention, the lowest detection limit is 100 copies / [mu] L of RNA, and compared with a conventional PCR method, the detection period is shortened, the detection steps are simplified, and the sensitivity is higher than that of the conventional PCR method.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid visualization detection, and in particular to the technical field of nucleic acid visualization detection of fever with thrombocytopenia syndrome. Background Art

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is an acute hemorrhagic febrile disease caused by Severe fever with thrombocytopenia syndrome virus (SFTSV). The main symptoms are fever, fatigue, nausea, vomiting and other gastrointestinal reactions. Clinical trials often show thrombocytopenia and leukocytopenia. Most patients have liver and kidney damage, and a few critically ill patients die from multiple organ failure. SFTSV, also known as Dabiebandavirus (DBV), belongs to the Bunyavirales order, Phenuiviridae family, and Bandavirus genus. The virus particles are spherical or ellipsoidal, with a diameter of about 80~100 nm, a double lipid envelope, and 5~10 nm long glycoprotein protrusions distributed on the surface. The viral genome is composed of three single-stranded negative-sense RNAs: large (L), medium (M), and small (S). The L segment encodes the RNA-dependent RNA polymerase (RdRp), which is involved in the RNA replication of the viral genome; the M segment encodes the membrane glycoprotein precursor (GPC), which is cleaved and modified to form two membrane proteins, Gn and Gc, which are expressed on the viral lipid bilayer envelope; the S segment encodes the viral nucleocapside protein (NP) and nonstructural protein (NS).

[0003] It is currently believed that SFTSV is an insect-borne virus, with ticks being the main vector. Humans are infected with SFTSV through tick bites, and the regular hosts of ticks include most mammals, including goats, cattle, donkeys, pigs, deer, cats, etc. Therefore, this mode of transmission causes most patients infected with SFTSV to come from rural areas such as mountainous areas or hills. SFTSV can also be transmitted from person to person, including contact with patient urine, feces, throat secretions, blood, respiratory secretions, and even aerosols.

[0004] Currently, there is no vaccine or targeted treatment for SFTSV, and only symptomatic treatment is provided. Therefore, early diagnosis of SFTSV is crucial for the treatment of the disease. The existing detection method is PCR detection, which has a long cycle, complex operation and low sensitivity. Therefore, it is urgent to establish a set of rapid, simple and accurate diagnostic methods to provide new detection technology and material basis for the rapid detection of fever with thrombocytopenia syndrome and the monitoring and early warning of pathogens. Summary of the invention

[0005] The present invention solves the problems of complex operation, low sensitivity and false positive results due to aerosol formation when detecting fever with thrombocytopenia syndrome virus in the prior art by providing a fever with thrombocytopenia syndrome virus nucleic acid detection primer and kit.

[0006] A primer set for detecting viral nucleic acid of fever with thrombocytopenia syndrome, comprising: FIP, BIP, F3, B3, LF and LB.

[0007] Further preferably, the FIP sequence is SEQ ID NO.1.

[0008] Further preferably, the BIP sequence is SEQ ID NO.2.

[0009] Further preferably, the F3 sequence is SEQ ID NO.3.

[0010] Further preferably, the B3 sequence is SEQ ID NO.4.

[0011] Further preferably, the LF sequence is SEQ ID NO.5.

[0012] Further preferably, the LB sequence is SEQ ID NO.6.

[0013] A kit for detecting nucleic acid of fever with thrombocytopenia syndrome virus, comprising: the above-mentioned primer set, 50 μL of S fragment positive plasmid, 250 μL of 10× polymerase buffer, 100 μL of Bst2.0WarmStart®DNA polymerase, 50 μL of AMV enzyme, 10 mM dNTP, 350 μL of betaine solution with a final concentration of 0.2 M, 200 μL of magnesium sulfate solution with a final concentration of 4 mM, and a disposable closed nucleic acid visualization detection device.

[0014] Further preferably, the primer addition amount is 100 μL each for FIP and BIP, 25 μL each for F3 and B3, and 50 μL each for LF and LB.

[0015] Further preferably, the reaction temperature of the kit is 63° C. and the reaction time is 50 min.

[0016] Beneficial Effects (1) The primer set and kit described in the present invention have high specificity in detecting the fever with thrombocytopenia syndrome virus. Through preliminary analysis and comparison of the fever with thrombocytopenia syndrome virus gene sequence, the conserved region of the S segment gene was screened as the target. The target sequence is highly conserved in the fever with thrombocytopenia syndrome virus gene and is different from the Heartland bandavirus (HRTV) of the genus Bandavirus of the family Phleboviridae. According to the base sequence of the S fragment of the fever with thrombocytopenia syndrome virus, an identification detection primer set was designed, and the primers used in the present invention were verified by specific evaluation to have no cross reaction with other viruses that cause similar clinical symptoms, such as Rift Valley fever phlebovirus (RVFV), Crimean-Congo hemorrhagic fever virus (CCHFV), influenza A virus H1N1 (Influenza A virus H1N1), Japanese encephalitis virus (Japanese encephalitis virus, JEV), and dengue virus (Dengue virus, DENV), so the detection accuracy is high.

[0017] (2) The kit of the present invention is used in detection, and the minimum detection limit is 100 copies / μL RNA. Compared with the conventional PCR method, it not only shortens the detection cycle, but also simplifies the detection steps and has a higher sensitivity.

[0018] (3) The kit of the present invention combines loop-mediated isothermal amplification technology with closed colloidal gold immunochromatographic test strips, which gets rid of the dependence of traditional nucleic acid detection methods on precision temperature-changing equipment. Effective amplification of the target gene of the fever with thrombocytopenia syndrome virus can be achieved under constant temperature conditions. In addition, the interpretation of the amplification results by a fully enclosed nucleic acid rapid detection device can not only realize the visualization of the results, but also avoid false positive results caused by aerosols. The test results are stable and reliable, and it is expected to realize rapid visualization detection at the epidemic site.

[0019] (4) The primer set described in the present invention provides a new material basis and detection technology for the rapid detection of fever with thrombocytopenia syndrome and the monitoring and early warning of pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The figure shows the detection results of RT-LAMP at different amplification reaction temperatures, where Negative is a negative control; Figure 2 The figure shows the detection results of RT-LAMP with different amplification reaction times, where Negative is the negative control; Figure 3 This is the test result of RT-LAMP sensitivity, where Negative is the negative control; Figure 4 This is the result of RT-LAMP specificity detection, where Negative is the negative control. DETAILED DESCRIPTION

[0021] Example 1. Establishment of RT-LAMP detection method for fever with thrombocytopenia syndrome virus.

[0022] 1. Design and synthesis of primers and probes.

[0023] The S segment gene sequences of 40 strains of FTSV, including existing variants, isolated from different countries, regions and times, were compared using the software MEGA7. The comparison results were then combined with the literature and commonly used laboratory detection targets to determine the nucleoprotein (NP) gene as the target gene for detecting FTSV, and the conserved region of the nucleoprotein was determined. Considering that the genes of the members of the genus Banyang virus have certain homology, the conserved region of the nucleoprotein was analyzed and compared with Heartland bandavirus (HRTV), and finally the nucleotide sequence 1483-1707 located on the S segment base sequence of FTSV was determined as the target for detecting FTSV to ensure the specificity of the primer set. Then, according to the LAMP primer design principle, two pairs of primer combinations were designed and synthesized, and primer sets with high amplification efficiency were screened out.

[0024] The primers used in the universal detection method for the NF-CP virus NP gene include: The inner primer FIP sequence is shown in SEQ ID NO.1: 5'-AGCTGAAGGAGACAGGTGGAGACCTCGAGTCAGGGCAAAGA-3'; The inner primer BIP sequence is shown in SEQ ID NO.2: 5'-GGATCAAGGCCTTCATAGGCCACAGTGGAGTTTGGTGAGCAG-3'; The sequence of the outer primer F3 is shown in SEQ ID NO.3: 5′-CCCTGATGCCTTGACGATC-3′; The sequence of the outer primer B3 is shown in SEQ ID NO.4: 5′-GTCAGAGTGGTCCAGGATTG-3′; The loop primer LF sequence is shown in SEQ ID NO.5: 5'-[Biotin]GCTCTCTCGCGAAATCCTCA-3'; The sequence of loop primer LB is shown in SEQ ID NO.6: 5'-[FAM]GGGTGAAGGATACTAAGTTCATCAT-3'.

[0025] The kit also provides a full-length positive plasmid of the S segment gene of the fever with thrombocytopenia syndrome virus, and the sequence of the S segment is shown in SEQ ID NO.7.

[0026] Note: FAM: carboxyfluorescein; Biotin: biotin.

[0027] 2. Establishment of RT-LAMP detection system.

[0028] The reaction system was 25 μL, which included: 0.5 μL each of 10 μM inner primers FIP and BIP, 0.125 μL each of 10 μM outer primers F3 and B3, 0.25 μL each of 10 μM loop primers LF and LB, 2.5 μL of 10× polymerase buffer, 1 μL of Bst2.0 WarmStart® DNA polymerase, 0.5 μL of AMV enzyme, 3.5 μL of 10 mM dNTP, 3.5 μL of betaine solution with a final concentration of 0.2 M, 2 μL of RNA template, 8.25 μL of ddH2O, and 2 μL of magnesium sulfate solution with a final concentration of 4 mM. The above reagents were mixed in the reaction tube and placed in a constant temperature water bath for incubation. Then, the reaction tube was placed in a fully enclosed nucleic acid rapid detection device (Hangzhou Yousida Biotechnology Co., Ltd., Jiangsu, China) for detection.

[0029] The criteria for judging the test results are as follows: if red bands appear at both the quality control line and the test line by naked eye observation, the sample contains the target RNA and the amplification product is positive; if a red band appears at the quality control line but no band appears at the test line, the sample does not contain the target RNA and the amplification product is negative; if no red band appears at the quality control line, the test result is invalid.

[0030] 3. Optimization of RT-LAMP reaction temperature.

[0031] The positive plasmid containing the full-length S segment gene of HFTSV was used as the template for LAMP detection. 10 The positive plasmid of the full-length gene of the S segment of the fever with thrombocytopenia syndrome virus was diluted 10 times in series to 2.13×10 -1 The amplified products were detected by a disposable nucleic acid visualization detection device. The results showed that the minimum detection limit of the RT-LAMP method for the NP gene was 2.13 copies / μL under the conditions of 61-67 ℃, as shown in Figure 2. Figure 1 At this time, the reaction temperature is 63 ℃, which is the optimal reaction temperature.

[0032] 4. Optimization of RT-LAMP reaction time.

[0033] Similarly, the positive plasmid containing the full-length gene of the S segment of the fever and thrombocytopenia syndrome virus was used as the template for LAMP detection. 10 The positive plasmid of the full-length gene of the S segment of the HFTS virus was diluted to 2.13×10 copies / μL. -1 The amplified products were detected by a disposable nucleic acid visualization detection device. The results showed that the minimum detection limit of the RT-LAMP method for the NP gene was 2.13 copies / μL when amplified at 63°C. Figure 2 At this time, the reaction time is 50 min, that is, 50 min is the optimal reaction time.

[0034] Example 2. Evaluation of the sensitivity of the RT-LAMP detection method for HFTS virus.

[0035] The RNA fragment containing the target gene of the S fragment of the fever and thrombocytopenia syndrome virus synthesized by Bao Ri Yi Biotechnology (Beijing) Co., Ltd. was used as a detection template for evaluating the sensitivity of this method. For its specific sequence information, see SEQ ID NO.8.

[0036] The RNA fragments of the target gene of the S segment of the fever with thrombocytopenia syndrome virus were serially diluted 10 times, and the amplified products were detected by a disposable nucleic acid visualization detection device after amplification at 63 ° C for 50 min according to the RT-LAMP detection system established in Example 1. The results showed that after using the RNA of the target gene of the S segment of the fever with thrombocytopenia syndrome virus as a template, the sensitivity of the nucleic acid visualization detection method was 100 copies / μL, see Figure 3 .

[0037] Example 3. Evaluation of the specificity of the RT-LAMP detection method for HFTS virus.

[0038] Since viruses such as Rift Valley fever virus (RVFV), Crimean-Congo hemorrhagic fever virus (CCHFV), influenza A virus (H1N1), Japanese encephalitis virus (JEV), and dengue virus (DENV) can all cause fever symptoms in humans in the early stages, it is difficult to identify which pathogen is causing the infection based solely on clinical symptoms. RVFV and SFTSV are both members of the Phleboviridae family, and CCHFV and SFTSV are both members of the Bunyavirales order. The detection method for SFTSV should have the characteristic of not reacting non-specifically with other viruses to avoid misleading medical workers in the correct formulation of patient treatment plans. To this end, the present invention uses SFTSV (HBMC16_human_2015 strain RNA), RVFV (NP positive plasmid), CCHFV (RNA), H1N1 (WSN strain RNA), JEV (RNA) and DENV (RNA) as templates to evaluate the specificity of the RT-LAMP detection method for fever with thrombocytopenia syndrome virus. The results showed that the RT-LAMP method for the NP gene of the fever with thrombocytopenia syndrome virus only showed a positive result for the RNA extracted from the HBMC16_human_2015 strain of SFTSV, while RVFV (NP positive plasmid), CCHFV (RNA), H1N1 (WSN strain RNA), JEV (RNA) and DENV (RNA) all showed negative results, see Figure 4 .

[0039] The results show that the RT-LAMP detection method for fever with thrombocytopenia syndrome virus established by the present invention does not cross-react with other Phleboviridae viruses, other Phleboviridae viruses, or other viruses with similar early symptoms during the detection process, and has high specificity. The other Phleboviridae viruses such as RVFV, the other Bunyavirales viruses such as CCHFV, and the viruses with similar early symptoms such as H1N1, JEV, DENV and other viruses.

[0040] Example 4. Assembly of the RT-LAMP detection kit for fever with thrombocytopenia syndrome virus.

[0041] 100 μL each of inner primers FIP and BIP, 25 μL each of outer primers F3 and B3, 50 μL each of loop primers LF and LB, 50 μL of S fragment positive plasmid, 250 μL of 10× polymerase buffer, 100 μL of Bst2.0 WarmStart® DNA polymerase, 50 μL of AMV enzyme, 10 mM dNTP, 350 μL of betaine solution with a final concentration of 0.2 M, 200 μL of magnesium sulfate solution with a final concentration of 4 mM and a disposable closed nucleic acid visualization detection device were assembled into a 96T (96 test) fever with thrombocytopenia syndrome virus RT-LAMP detection method kit.

Claims

1. A primer set for detecting nucleic acid of fever with thrombocytopenia syndrome virus, characterized in that: The primer set includes: FIP, BIP, F3, B3, LF and LB.

2. The primer set according to claim 1, characterized in that: The FIP sequence is as SEQ ID NO.

1.

3. The primer set according to claim 1, characterized in that: The BIP sequence is as SEQ ID NO.

2.

4. The primer set according to claim 1, characterized in that: The F3 sequence is as SEQ ID NO.

3.

5. The primer set according to claim 1, characterized in that: The B3 sequence is as SEQ ID NO.

4.

6. The primer set according to claim 1, characterized in that: The LF sequence is as shown in SEQ ID NO.

5.

7. The primer set according to claim 1, characterized in that: The LB sequence is as SEQ ID NO.

6.

8. A kit for detecting nucleic acid of fever with thrombocytopenia syndrome virus, characterized in that: The kit comprises: the primer set according to any one of claims 1 to 7, 50 μL of S fragment positive plasmid, 250 μL of 10× polymerase buffer, 100 μL of Bst2.0 WarmStart® DNA polymerase, 50 μL of AMV enzyme, 10 mM dNTP, 350 μL of betaine solution with a final concentration of 0.2 M, 200 μL of magnesium sulfate solution with a final concentration of 4 mM, and a disposable closed nucleic acid visualization detection device.

9. The kit according to claim 8, characterized in that: The primers added were 100 μL each for FIP and BIP, 25 μL each for F3 and B3, and 50 μL each for LF and LB.

10. The kit according to claim 8, characterized in that: The reaction temperature of the kit is 63°C and the reaction time is 50 min.

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