Novel highly sensitive nucleic acid detection method for Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV, Bandavirus dabieense)

Abstract

This invention provides an RT-PCR primer set for nucleic acid amplification derived from Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) and a method for detecting SFTSV. [Solution] An RT-PCR primer set for SFTSV-derived nucleic acid amplification, comprising a primer set comprising a forward primer consisting of a specific first nucleotide sequence or a nucleotide sequence having 80% or more identity with the first nucleotide sequence and annealing with the complementary strand of the first nucleotide sequence under RT-PCR conditions, a reverse primer consisting of a specific second nucleotide sequence or a nucleotide sequence having 80% or more identity with the second nucleotide sequence and annealing with the complementary strand of the second nucleotide sequence under RT-PCR conditions, and a probe consisting of a specific third nucleotide sequence or a nucleotide sequence having 80% or more identity with the third nucleotide sequence and annealing with the complementary strand of the third nucleotide sequence, wherein the set includes at least one primer set selected from three groups of primer sets.

Description

[Technical Field]

[0001] The present invention relates to a novel primer set for detecting the nucleic acid of severe fever with thrombocytopenia syndrome virus with high sensitivity, a kit for detecting the virus, and a method for detecting the virus. [Background Art]

[0002] In recent years, in Japan, domestic cases of infection associated with pathogens identified overseas have been confirmed, such as the occurrence of domestic dengue virus infection cases. Due to the international movement of people, not only are emerging and re-emerging infectious diseases accompanying the discovery of new pathogens spreading domestically, but the risk of these pathogens being introduced into blood products such as donated blood is also increasing.

[0003] Among these, Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high fatality rate, which is mainly caused by infection with Severe fever with thrombocytopenia syndrome virus (SFTSV, Bandavirus dabieense) via ticks. Since domestic infection of this infectious disease has already been confirmed, the development of a screening method that can quickly detect the contamination of blood and reduce the risk of the virus being introduced into blood products is urgently needed.

[0004] In Japan, the identification of SFTSV is carried out by regional public health institutes. SFTSV identification has been performed by a method of detecting SFTSV-derived nucleic acid (genome) via conventional PCR using a primer set provided by the National Institute of Infectious Diseases (Non-Patent Documents 1 and 2).

[0005] However, the above conventional method does not have sufficient sensitivity to specifically detect SFTSV contaminated in blood, and thus has the problem that it cannot evaluate the risk of SFTSV contamination in blood products. In addition, conventional PCR commonly used in testing requires electrophoresis after the PCR reaction, which results in complicated procedures and also has the problem of poor quantitativity.

[0006] Therefore, in order to solve the above problems, a method for detecting SFTSV using real-time RT-PCR has been reported (Non-Patent Documents 3 and 4). SFTSV has three single-stranded RNAs consisting of S, M, and L segments as its genome, and the forward primers, reverse primers, and probe sequences for each genome were determined in the aforementioned documents. Currently, it is common to identify SFTSV using the method described in Non-Patent Document 3. However, this existing method has the problem that the primers and probes used in RT-PCR have not been optimized, and the detection of some SFTSVs is unstable. In addition, in the existing technology, each probe for the S, M, and L segments is modified with a different dye, and although information on which segment-derived nucleic acid is present in the sample can be obtained, the detection sensitivity is low, which is also a problem as it makes it unsuitable for use in blood products. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] IASR Vol. 35 p. 40-41: February 2014 issue [Non-Patent Document 2] Yu XJ, et al., N Engl J Med 364: 1523-1532, 2011 [Non-Patent Document 3] Yoshikawa, et al., J Clin Microbiol 52 (9):3325-3333, 2014 [Non-Patent Document 4] N. Shinohara, et al., Vox Sanguinis 109:417-419, 2015 [Overview of the project] [Problems that the invention aims to solve]

[0008] This invention was made in view of solving the above-mentioned problems, and aims to provide a novel SFTSV detection method with high detection sensitivity and specificity for SFTSV. Furthermore, it aims to establish a highly sensitive nucleic acid testing method for SFTSV, thereby contributing to ensuring the safety and stable supply of blood products. [Means for solving the problem]

[0009] As a result of diligent research, the inventors have found optimal primer and probe sequences that simultaneously achieve high detection sensitivity for the SFTSV genome and avoid nonspecific reactions to related viruses, thereby establishing a novel and highly sensitive SFTSV nucleic acid detection method using RT-PCR and completing the present invention. That is, the present invention includes the following aspects.

[0010] Section 1. A forward primer consisting of the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 1, and which anneals to the complementary strand of the nucleotide sequence of SEQ ID NO: 1 under RT-PCR conditions. A reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 2, and which anneals to the complementary strand of the nucleotide sequence of SEQ ID NO: 2 under RT-PCR conditions, and A probe consisting of the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 3, and which anneals to the complementary strand of the SEQ ID NO: 3 nucleotide sequence under RT-PCR conditions. Primer set S consisting of, A forward primer consisting of the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 4, and which anneals to the complementary strand of the nucleotide sequence of SEQ ID NO: 4 under RT-PCR conditions. A reverse primer consisting of the nucleotide sequence of SEQ ID NO: 5, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 5, and which anneals to the complementary strand of the nucleotide sequence of SEQ ID NO: 5 under RT-PCR conditions, and A probe consisting of the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 6, and which anneals to the complementary strand of the SEQ ID NO: 6 nucleotide sequence under RT-PCR conditions. Primer set M consisting of and A forward primer consisting of the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 7, and which anneals to the complementary strand of the nucleotide sequence of SEQ ID NO: 7 under RT-PCR conditions. A reverse primer consisting of the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 8, and which anneals to the complementary strand of the nucleotide sequence of SEQ ID NO: 8 under RT-PCR conditions, and A probe consisting of the nucleotide sequence of SEQ ID NO: 9, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 9, and which anneals to the complementary strand of the SEQ ID NO: 9 nucleotide sequence under RT-PCR conditions. Primer set L consisting of A set of RT-PCR primers for nucleic acid amplification derived from severe fever with thrombocytopenia syndrome virus (Bandavirus dabieense, SFTSV), comprising at least one primer set selected from the group consisting of the following. Section 2. The RT-PCR primer set according to claim 1, comprising at least two primer sets selected from the group consisting of primer sets S, M, and L. Section 3. The RT-PCR primer set according to item 1 or 2, comprising the primer set S. Section 4. An RT-PCR primer set according to any one of claims 1 to 3, comprising the primer sets S, M, and L. Section 5. A set of RT-PCR primers described in any one of items 1 to 4 for amplification of SFTSV-derived nucleic acids having genotypes J1, J2, J3, C3, C4, and C5. Section 6. The RT-PCR primer set according to any one of items 1 to 5, which does not detect nucleic acids derived from Hazara virus, Nipah virus, Heartland virus, Mobala virus, Mopeia virus, Rift Valley fever virus, Issyk-kul virus and Soft tick bunyavirus by RT-PCR. Item 7. The RT-PCR primer set according to any one of items 1 to 6, which is for testing blood products, donated blood serving as a raw material thereof, and raw material plasma. Item 8. A kit for detecting SFTSV, comprising the RT-PCR primer set according to any one of items 1 to 7. Item 9. a step of performing RT-PCR using the RT-PCR primer set according to any one of items 1 to 7 or the kit according to claim 8, with SFTSV-derived genomic RNA as a template A method for detecting SFTSV, comprising the above step. [Advantageous Effects of Invention]

[0011] Compared with conventional techniques, the SFTSV detection method of the present invention has higher detection rate, higher detection sensitivity, and higher specificity. [Brief Description of Drawings]

[0012] [Figure 1] Fig. 1 is a diagram showing the working process for constructing the novel nucleic acid detection method of the present invention. [Figure 2](A) is a diagram showing the genomic structure of SFTSV and the screening procedure for the designed nucleic acid detection primers. (B) is a diagram showing the results of primary screening of primers against the S segment of SFTSV strain J1 using SYBR Green. (C) is a diagram showing the results of primary screening of primers against the M segment of SFTSV strain J1 using SYBR Green. (D) is a diagram showing the results of primary screening of primers against the L segment of SFTSV strain J1 using SYBR Green. (E) is a diagram showing the results of rescreening performed using RNAs derived from SFTSV strains J2 and J3. [Figure 3] (A) is a diagram showing the results of secondary screening of TaqMan probes using RNAs derived from strains J1 to J3. (B) is a diagram showing the results of secondary screening of TaqMan probes using RNAs derived from strains C1 to C5. [Figure 4] It is a diagram showing the results of a false positive test for samples from SFTSV-uninfected individuals in secondary screening. [Figure 5] It is a diagram comparing the detection sensitivity and detection rate of Example 1 of the present application and a comparative example. [Figure 6] It is a diagram showing the significance test of Example 1 of the present application and a comparative example. [Figure 7] It is a diagram showing waveforms when clinical specimens are measured using Example 1 of the present application and a comparative example. [Figure 8] (A) is a diagram showing the waveform of strain J1 (18v4-8) in FIG. 7. (B) is a diagram showing the waveform of strain JC5 (21v4a-2) in FIG. 7. [Figure 9] It is a diagram showing a waveform when a Rickettsia japonica positive sample is used. [Figure 10] It is a diagram showing the results of a specificity test using related viruses.

Mode for Carrying Out the Invention

[0013] Severe fever with thrombocytopenia syndrome virus (SFTSV) is a virus identified as the pathogen of severe fever with thrombocytopenia syndrome (SFTS), a viral hemorrhagic fever. Its genome consists of single-stranded RNA and is segmented into three segments: L (Large), M (Medium), and S (Small).

[0014] The present invention relates to RT-PCR (reverse transcription PCR), which involves reverse transcribing SFTSV-derived genomic RNA into cDNA (complementary DNA) and amplifying SFTSV-derived nucleic acid using PCR with the resulting cDNA as a template. By detecting the amplified nucleic acid, it is possible to determine whether or not SFTSV is present in the sample. The RT-PCR of the present invention may also be quantitative PCR, such as real-time RT-PCR. By using real-time PCR, the amplified nucleic acid can be detected without the need for electrophoresis. Therefore, real-time PCR is advantageous because it can provide a simple and rapid testing method for determining the presence or absence of SFTSV in a sample.

[0015] The first embodiment of the present invention is a primer set (RT-PCR primer set) used for RT-PCR for nucleic acid amplification of SFTSV. The primer set used for RT-PCR includes forward primers, reverse primers, and a fluorescently labeled probe. All of these oligonucleotides can be synthesized by conventional methods such as chemical synthesis using an automated DNA synthesizer. In the present invention, primers were designed for the entirety of the three genomes of SFTSV, and a primer set capable of detecting SFTSV with high sensitivity was found by comprehensively screening them. Furthermore, the primer set of the present invention can detect multiple genotypes of SFTSV, which is advantageous compared to conventional techniques. Moreover, since it does not detect nucleic acids of related viruses, it can specifically detect SFTSV.

[0016] This invention provides a method for detecting target SFTSV-derived nucleic acids using a fluorescently labeled probe (5'-nuclease method). The fluorescently labeled probe used in this invention (hereinafter sometimes simply referred to as "probe") has one end of its base sequence modified with a fluorescent substance and the other end modified with a quencher substance that suppresses fluorescence generation. Examples of such probes include TaqMan probes, with TaqMan MGB probes being preferred. SFTSV is detected by detecting the fluorescent dye released from the probe. The detection method is not particularly limited as long as it can detect fluorescence from the probe, but a real-time PCR device is preferred.

[0017] The fluorescent dyes used in the probes are not particularly limited, but examples include fluorothane (FAM) and Cy5. The quenchers used with the probes are selected to correspond to the fluorescent dyes used. The fluorescent dyes of the probes included in the primer sets S, M, and L described later may be the same or different. When the probes included in primer sets S, M, and L are modified with the same fluorescent dye, detection sensitivity can be increased by using multiple primer sets. Therefore, in this invention, it is preferable that the probes for the three genomes are modified with the same fluorescent dye.

[0018] The forward primers, reverse primers, and probes included in Primer Set S are complementary strands of a portion of the template cDNA synthesized from the S segment of the SFTSV genome. They specifically anneal to the S segment of the template cDNA to amplify SFTSV-derived nucleic acids. Therefore, Primer Set S can be used to determine whether or not SFTSV is present in a sample, and more specifically, to detect whether or not an S segment derived from the SFTSV genome is present.

[0019] Similarly, the oligonucleotides contained in primer sets M and L specifically anneal to template cDNA synthesized from the M and L segments of the SFTSV genome, respectively, to amplify SFTSV-derived nucleic acids. Therefore, by using these primer sets, it is possible to determine whether or not SFTSV is present in a sample, and more specifically, to specifically detect whether or not the M or L segment of SFTSV is present in the sample.

[0020] There are no particular restrictions on the samples used in the present invention, and purified SFTSV genomic RNA, SFTSV suspension, SFTSV-infected cell suspension, and clinical specimens from SFTSV-infected patients can be used. If necessary, genomic RNA can be extracted from the sample and then reverse transcribed to obtain cDNA to be used as a template for RT-PCR. The method for obtaining the template cDNA is not particularly limited as long as sufficient purity and quantity of cDNA can be obtained to detect SFTSV-derived nucleic acids by RT-PCR, and conventional methods or commercially available preparation kits can be used. RNA extraction and reverse transcription from the sample may be performed in separate steps or in a single step. Alternatively, the extracted RNA may not be pre-converted into cDNA, but instead added to the RT-PCR sample as a template, and cDNA conversion and RT-PCR may be performed in a single step. The SFTSV detection method of the present invention shows higher detection rates and sensitivity compared to conventional techniques for both purified SFTSV and clinical specimens from SFTSV-infected patients.

[0021] These primer sets S, M, and L may be used individually or in combination. Preferably, the set contains at least two of the primer sets S, M, and L. More preferably, it contains primer set S. Most preferably, it contains primer sets S, M, and L. Whether primer sets S, M, and L are used individually or in combination, nucleic acids derived from SFTSV in the sample can be detected in either case.

[0022] The oligonucleotides described above, having 80% or more homology to the base sequences shown in SEQ ID NOs: 1 to 9, and annealing to template cDNA under RT-PCR conditions, can be used to amplify SFTSV-derived nucleic acids and thus be used for SFTSV detection. In the above, the homology is preferably 90% or more, and more preferably 95% or more. RT-PCR conditions refer to the annealing conditions in RT-PCR, which are usually set to a temperature 2 to 3°C lower than the primer's Tm value. The annealing conditions in RT-PCR are typically 45 to 60°C and 10 to 60 seconds.

[0023] The temperature at which these oligonucleotides anneal to the template cDNA is 57°C to 63°C, preferably 60°C. Therefore, preferred RT-PCR conditions in the present invention are, for example, 1) 50°C for 20 minutes, 2) 95°C for 15 minutes, 3) 94°C for 45 seconds, 4) 60°C for 45 seconds, and repeating steps 3) and 4) 45 times.

[0024] Furthermore, the oligonucleotides of the present invention, as long as they anneal to template cDNA at the aforementioned temperature and amplify nucleic acids derived from SFTSV, also include oligonucleotides represented by the base sequences described in SEQ ID NOs. 1 to 9 that have undergone mutations or modifications such as deletion, insertion, or substitution of one to several bases (5, 4, 3, or 2).

[0025] The primer set amplifies nucleic acids derived from SFTSV with genotypes J1, J2, J3, C3, C4, and C5. Therefore, the present invention can detect multiple genotypes of SFTSV and has superior detection sensitivity compared to conventional techniques in which the detection of some genotypes, such as the J3 strain, is unstable.

[0026] Conversely, when using the primer set of the present invention, nucleic acids derived from SFTSV-related viruses such as Hazara virus, Nipah virus, Heartland virus, Mobala virus, Mopeia virus, Rift Valley fever virus, Issyk-kul virus, and Soft tick bunyavirus are not detected by RT-PCR. In other words, the primer set of the present invention is an excellent SFTSV detection method that specifically detects SFTSV and has a low false-positive rate.

[0027] The primer set of the present invention can detect SFTSV at lower concentrations compared to conventionally used primer sets. In other words, it has higher detection sensitivity compared to existing methods. Since a test with higher detection sensitivity is necessary to ensure the safety of blood products, the present invention can be used to determine whether or not SFTSV is present in raw materials such as blood products and donated blood.

[0028] A second embodiment of the present invention is an SFTSV detection kit comprising the primer set. The kit can be used as a method for detecting SFTSV of multiple genotypes. In addition to the primer set, the kit includes reagents necessary for performing RT-PCR as needed. Examples of such reagents include buffers, nucleic acid polymerases (DNA polymerase, RNA polymerase, reverse transcriptase, etc.), enzyme substrates (dNTPs, etc.), and labeled detection substances. The kit of the present invention may also include a positive control to confirm that the detection reaction is possible with the primer set of the present invention. An example of a positive control is a DNA fragment containing the region amplified by the primer set of the present invention.

[0029] A third embodiment of the present invention is an SFTSV detection method comprising the step of performing RT-PCR using the primer set or kit with SFTSV-derived genomic RNA as a template. After the above step, the method may also include a step of detecting whether or not SFTSV is present in the sample by detecting the amplified nucleic acid. The device for detecting the amplified nucleic acid is not particularly limited as long as it can detect the fluorescent dye derived from the probe, but a real-time PCR device is preferred. [Examples]

[0030] The present invention is further illustrated by the following embodiments, which should not be construed as further limitations.

[0031] 1. Screening of primer sets The primer set of the present invention can be obtained through the following steps. 1) Based on sequence information from various clinical viral isolates, primers were comprehensively designed for the entire SFTSV genome based on the most frequently occurring nucleotide sequences, and a large-scale primer library was constructed. Primer3plus software was used for primer design. The number of forward and reverse primer combinations created was 80, 110, and 160 sets for the S, M, and L segments, respectively (Figure 2 (A)). 2) Primary screening was performed by using genomic RNA extracted from clinical isolates (J1 strain) as a template and subjecting the primer library designed in 1. above to real-time RT-PCR. For detection, SYBR Green, which intercalates into double-stranded DNA and emits fluorescence, was used. One million copies of a mixture of viral RNA from four J1 strains (SPL30, SPL067, SPL070, and SPL120) were used as the template. The PCR conditions were (1) 50°C for 20 minutes, (2) 95°C for 15 minutes, (3) 94°C for 15 seconds, (4) 60°C for 20-30 seconds, and (5) 72°C for 30 seconds. Steps (3), (4), and (5) were repeated 35-45 times. 3) Based on the Ct values ​​and melting curves described in 2) above, detection sensitivity and specificity were compared, and 23 primer sets (Figure 2 (B)), 28 sets (Figure 2 (C)), and 45 sets (Figure 2 (D)) with superior characteristics were selected for the S, M, and L segments, respectively. 4) The 96 primer sets selected in step 3) above were re-screened using the J2 and J3 strains in the same manner. 24 primer sets were efficient with the J2 strain (Figure 2 (E), upper bar graph with fill and dashed lines), and 45 primer sets were efficient with the J3 strain (Figure 2 (E), lower bar graph with fill and dashed lines). Of these, 3, 3, and 9 sets were selected for the S, M, and L segments, respectively, as primer sets that showed high detection sensitivity and specificity in both the J2 and J3 strains (Figure 2 (E), total of 15 sets (dashed bar graph)). 5) TaqMan MGB probes were designed using the Primer Express software for the regions amplified by each primer selected in 4) above. Specific probes could not be designed for one set of primers each for S, M, and L segments. Excluding that one set, one probe was designed for each primer set, resulting in a total of two probes for S segments (S-60 and S-62), two for M segments (M-47 and M-87), and eight for L segments (L-35, L-67, L-84, L-92, L-115, L-128, L-132, and L-150). In selecting the probes, probes with many mutations were excluded based on the results of multiplex sequence alignment. FAM was used for all probe fluorescent dyes. 6) Using purified viral RNA from a total of 13 strains shown in Table 1—four J1 strains (SPL30, SPL067, SPL070, and SPL120), two J2 strains (SPL057 and SPL100), two J3 strains (SPL004 and SPL230), one C3 strain (HB29), two C4 strains (SPL179 and SPL193), and two C5 strains (SPL087 and SPL238)—as templates, the TaqMan probes designed and prepared in 5) were subjected to real-time RT-PCR for secondary screening. The template RNA concentration was 1 x 10⁻¹⁶. 5The results were copies / well. The PCR conditions were (1) 50°C for 20 minutes, (2) 95°C for 15 minutes, (3) 94°C for 45 seconds, and (4) 60°C for 45 seconds. Steps (3) and (4) were repeated 45 times. The results are shown in Figure 3. 7) Using plasma from SFTSV-free individuals as a template, the TaqMan probe designed and prepared in 5) was subjected to real-time RT-PCR to examine for false positives. The PCR conditions were (1) 50°C for 20 minutes, (2) 95°C for 15 minutes, (3) 94°C for 45 seconds, and (4) 60°C for 45 seconds. Steps (3) and (4) were repeated 45 times. The results are shown in Figure 4. Nucleic acid amplification was observed and a false positive was observed only when using the primer and probe set with probe name S-62. 8) For each segment, S-60 was selected for the S segment and M-87 for the M segment as primer-probe combinations (primer sets) that did not react with SFTSV-uninfected samples and showed excellent amplification efficiency only with purified SFTSV RNA. For the L segment, L-35, L-67, L-115, and L-150 were selected, with L-115 being the most preferred combination among them. The most preferred S-60, M-87, and L-115 for each segment were selected and are shown in SEQ ID NOs. 1-9 in Table 2. The results of the detection specificity and detection sensitivity compared with existing methods are shown below.

[0032] [Table 1]

[0033] [Table 2]

[0034] 2. Comparison of detection sensitivity using SFTSV virus panel In this verification, the primers and probes shown in Table 2 were used. The primer sets S, M, and L of the present invention obtained in the screening described in 1. above refer to primer sets consisting of SEQ ID NOs: 1-3, 4-6, and 7-9, respectively. Example 1 (KMU) included all primers and probes of SEQ ID NOs: 1-9. Similarly, Comparative Example 1 (JRC) included all primers and probes of SEQ ID NOs: 10-18, and Comparative Example 2 (V1) included all primers and probes of SEQ ID NOs: 19-31. The detection sensitivity was compared using the SFTSV virus panel in Table 1. The forward and reverse primer sequences of Comparative Example 1 and Comparative Example 2 were identical. The fluorescent dye used for the probes was FAM in both cases.

[0035] For Example 1 (KMU) and Comparative Example 1 (JRC), nine primers and probes for three segments were added to the reaction mixture simultaneously. For Comparative Example 2 (V1), thirteen primers and probes for three segments were added simultaneously. The detection sensitivity for a panel of 13 SFTSV virus strains was compared using multiplex real-time RT-PCR. Purified RNA from a total of 13 strains, as shown in Table 1, was used as the template. The PCR conditions were (1) 50°C for 20 minutes, (2) 95°C for 15 minutes, (3) 94°C for 45 seconds, and (4) 60°C for 45 seconds. Steps (3) and (4) were repeated 45 times. In this validation, a Ct value of less than 45 was considered positive (virus RNA was detected).

[0036] Each virus strain was measured eight times in a sample size of N=2, and the average Ct values ​​for N=2 are shown in Table 3. When calculating the average value, the Ct values ​​for 1 well of Example 1, 3 wells of Comparative Example 1, and 50 wells of Comparative Example 2, which were below the detection limit, were considered to be 45, the maximum number of RT-PCR cycles, and the average value for N=2 was calculated accordingly. The number of detections where the Ct value was less than 45 out of a total of 16 tests for each virus strain is shown in the right table of Figure 5, and the percentage of tests where the Ct value was less than 45 (detection rate) out of the total number of tests (13 strains x 16 tests) is shown in the left graph of Figure 5. Example 1 of this application had the highest detection rate among the three. Furthermore, in Comparative Example 2, which is currently used for SFTSV detection, the detection rate was particularly low for certain strains such as strain J3 (SPL004), suggesting that detection may not be possible with existing technology. In contrast, Example 1 of this application showed a significantly higher detection rate than Comparative Example 2. Therefore, it has been shown that the present invention is also effective for detecting SFTSV with genotypes that are difficult to detect with existing technology.

[0037] [Table 3]

[0038] As described above, the present invention is capable of detecting SFTSV of multiple genotypes. Furthermore, the present invention has higher detection sensitivity compared to the prior art. Therefore, it is considered useful for screening whether SFTSV is present in blood products and raw materials such as donated blood and raw plasma, i.e., for testing blood products and the donated blood and raw plasma that serve as their raw materials.

[0039] To verify the significant difference between Example 1 (KMU) and Comparative Examples 1 (JRC) and 2 (V1), the mean values ​​of N=2 shown in Table 3 were plotted in Figure 6. A significance test (Friedman test and a post-hoc Wilcoxon signed-rank test with Bonferroni correction) was performed on the Ct values ​​in Figure 6 using Prism10. The results showed that p<0.0001 was obtained for both Example 1 and Comparative Example 1, and for Example 1 and Comparative Example 2, indicating that Example 1 has significantly higher sensitivity than Comparative Examples 1 and 2.

[0040] 3. Verification using RNA from clinical specimens 3.1 Detection sensitivity using J1 strain and C5 strain The detection sensitivity of primer sets in Example 1 and the Comparative Example was compared using RNA extracted from clinical isolates (J1 and C5 strains) derived from SFTS patients. All samples were plasma, and RNA was extracted from 200 μL of each plasma sample into 50 μL of buffer using magLEAD 12gC from PSS (Precision System Science Co., Ltd.), which was used as a template for real-time RT-PCR. Table 4 shows the origin of the clinical samples used, the identified pathogen, and the copy number of the sample used as a template. The copy number of the sample was calculated by creating a calibration curve using the J1 strain (SPL030) with a known concentration. SFTSV was detected for each sample using Example 1 and the Comparative Example, and the results are shown in Figure 7.

[0041] [Table 4]

[0042] In Comparative Examples 1 and 2, as shown in the brackets on the right side of the figures, the signal tended to plateau at a lower fluorescence intensity than in Example 1 of the present invention when the sample had a low copy number (V1 and JRC in Figure 7). On the other hand, in Example 1 of the present invention, the fluorescence intensity did not plateau easily even when the sample had a low copy number (KMU in Figure 7). From this, it can be said that the present invention can stably obtain a high signal even in samples with a low copy number, and that its performance is better than existing detection systems.

[0043] In this invention, since the fluorescence intensity does not plateau even in low-copy-number samples, it is considered to have the advantage of making it easier to distinguish between SFTSV-derived signals and nonspecific signals even in samples where nonspecific reactions appear. Due to this advantage, this invention is considered to be applicable to risk assessment of SFTSV contamination in blood products.

[0044] Figure 8 shows representative waveforms during the detection of strain J1 (18v4-8) (Figure 8(A)) and strain C5 (21v4a-2) (Figure 8(B)). The horizontal axis represents the number of PCR cycles, and the vertical axis represents the fluorescence intensity. From the upper part of Figure 8, it was found that in the detection of strain J1, Example 1 of this application (solid line) had higher detection sensitivity than Comparative Example 2 (dashed line). Comparative Example 1 (dotted line) and Example 1 of this application showed similar reactivity. From Figure 8(B), it was found that in the detection of strain C5, Example 1 of this application had superior sensitivity compared to both Comparative Examples 1 and 2. As described above, it was found that the present invention has a higher detection rate and detection sensitivity compared to the conventional technology, not only for purified RNA but also for clinical samples derived from SFTSV-infected patients.

[0045] 3.2 Differential Diagnosis of Tick-borne Infections (False Positive Test) As a comparative experiment, to confirm that Rickettsia japonica, the pathogen of Japanese spotted fever which is transmitted by ticks like SFTS, is not detected by the present invention, verification by RT-PCR was performed using a Rickettsia japonica-positive sample in the same manner as in 3.1. As shown in Figure 9, it was confirmed that Rickettsia japonica was not detected in Example 1 of this application.

[0046] 3.3 Specificity Test RT-PCR was performed using purified RNA (the amount of RNA used was equivalent to the amount of virus shown in Figure 10) of SFTSV and related viruses such as Hazara virus, Nipah virus, Heartland virus, Mobala virus, Mopeia virus, Rift Valley fever virus, Issyk-kul virus, and Soft tick bunyavirus as templates, using primer sets S-60 alone, M-87 alone, L-115 alone, or Example 1 of this application (a three-complex, indicated as 3-plex in the figure) containing all three primer sets from Table 2. RT-PCR was also performed on strains J1, J2, and J3 as a control. No signals other than those from strains J1, J2, and J3 (SFTSV) were observed in any of the S-60, M-87, and L-115 sets, suggesting that nonspecific amplification was minimal (Figure 10). From these results, it was found that Example 1 of the present invention exhibits high specificity for SFTSV because no nonspecific reaction to related viruses was observed.

[0047] 3.4 Comparison of multi-plex and single-plex architectures As shown in Figure 10, in the amplification of strains J1, J2, and J3, sufficient nucleic acid amplification of SFTSV was observed even in single-plex PCR with each primer set S-60, M-87, and L-115 added individually. The present invention demonstrates that the present invention is a detection method that has high detection sensitivity and specificity for SFTSV even when using primer sets S-60, M-87, and L-115 individually. In other words, the present invention can be said to be a detection method that has high detection sensitivity and specificity for SFTSV even when using at least one primer set selected from the group consisting of primer sets S-60, M-87, and L-115. Furthermore, when using 3-plex PCR containing all of primer sets S-60, M-87, and L-115, a lower Ct value and higher final fluorescence intensity were observed compared to single-plex PCR. Therefore, it can be said that 3-plex PCR containing all of primer sets S, M, and L is a more sensitive detection method than single-plex PCR.

Claims

1. A forward primer consisting of the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 1, and which anneals to the complementary strand of the nucleotide sequence of SEQ ID NO: 1 under RT-PCR conditions. A reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 2, and which anneals to the complementary strand of the nucleotide sequence of SEQ ID NO: 2 under RT-PCR conditions, and A probe consisting of the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 3, and which anneals to the complementary strand of the SEQ ID NO: 3 nucleotide sequence under RT-PCR conditions. Primer set S consisting of, A forward primer consisting of the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 4, and which anneals to the complementary strand of the nucleotide sequence of SEQ ID NO: 4 under RT-PCR conditions. A reverse primer consisting of the nucleotide sequence of SEQ ID NO: 5, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 5, and which anneals to the complementary strand of the nucleotide sequence of SEQ ID NO: 5 under RT-PCR conditions, and A probe consisting of the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 6, and which anneals to the complementary strand of the SEQ ID NO: 6 nucleotide sequence under RT-PCR conditions. Primer set M consisting of and A forward primer consisting of the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 7, and which anneals to the complementary strand of the nucleotide sequence of SEQ ID NO: 7 under RT-PCR conditions. A reverse primer consisting of the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 8, and which anneals to the complementary strand of the nucleotide sequence of SEQ ID NO: 8 under RT-PCR conditions, and A probe consisting of the nucleotide sequence of SEQ ID NO: 9, or a nucleotide sequence having 80% or more identity with SEQ ID NO: 9, and which anneals to the complementary strand of the SEQ ID NO: 9 nucleotide sequence under RT-PCR conditions. Primer set L consisting of A set of RT-PCR primers for nucleic acid amplification derived from severe fever with thrombocytopenia syndrome virus (Bandavirus dabieense, SFTSV), comprising at least one primer set selected from the group consisting of the following.

2. The RT-PCR primer set according to claim 1, comprising at least two primer sets selected from the group consisting of primer sets S, M, and L.

3. The RT-PCR primer set according to claim 1, comprising the primer set S.

4. The RT-PCR primer set according to claim 1, comprising the primer sets S, M, and L.

5. The RT-PCR primer set according to claim 1 for amplification of SFTSV-derived nucleic acids having genotypes J1, J2, J3, C3, C4, and C5.

6. The RT-PCR primer set according to claim 1, wherein nucleic acids derived from Hazara virus, Nipah virus, Heartland virus, Mobala virus, Mopeia virus, Rift Valley fever virus, Issyk-kul virus, and Soft tick bunyavirus are not detected by RT-PCR.

7. The RT-PCR primer set according to claim 1, for use in testing blood products and donated blood and raw plasma used as raw materials for those products.

8. A kit for detecting SFTSV, comprising the RT-PCR primer set described in claim 1.

9. A step of performing RT-PCR using the RT-PCR primer set described in claim 1 or the kit described in claim 8, with SFTSV-derived genomic RNA as a template. A method for detecting SFTSV, including the method described above.

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