KR102238561B1 - Kit for diagnosing infection due to severe fever with thrombocytopenia syndrome virus - Google Patents

Abstract

The present invention relates to a kit for diagnosing Severe Fever with Thrombocytopenia Syndrome (SFTS) viral disease.

Description

Kit for diagnosing severe febrile thrombocytopenia syndrome (SFTS) viral infections {KIT FOR DIAGNOSING INFECTION DUE TO SEVERE FEVER WITH THROMBOCYTOPENIA SYNDROME VIRUS}

The present invention relates to a kit for diagnosing Severe Fever with Thrombocytopenia Syndrome (SFTS) viral disease.

Severe fever caused by Severe Fever with Thrombocytopenia Syndrome (SFTS) is an emerging viral disease common in China, Korea and Japan. There is currently no effective vaccine or specific treatment for SFTS. SFTS is a serious disease with symptoms such as high fever, vomiting, diarrhea, thrombocytopenia, leukocytosis and multiple organ failure, and a mortality rate of 6-30% (Yu XJ et al., N. Engl. J. Med. 2011; 364:1523-32; Ding F et al Clin Infect Dis 2013; 56: 1682-3).

In addition, seroconversion and viremia of the SFTS virus have been found in breeding animals such as goats, sheep, cattle, pigs and dogs, and these animals are also thought to act as intermediate mediators in the area where the SFTS virus is spread (Zhao L et al. Emerg Infect Dis 2013; 18: 963-5; Niu G et al. Emerg Infect Dis 2013; 19: 756-63).

In 2015, the Korea Centers for Disease Control and Prevention reported that doctors and nurses at university hospitals who were caring for patients infected with the severe febrile thrombocytopenia syndrome (SFTS) virus were secondarily infected with the virus. From 2013 when the first infected person was diagnosed to July 2015, there were a total of 122 confirmed patients with SFTS in Korea, of which 40 are known to have died.

Meanwhile, Chinese Laid-Open Patent Publication No. 102070704 discloses an SFTS virus amplification and detection kit using an SFTS virus.

However, the present inventors have devised an effective diagnostic kit for SFTS of a sequence capable of detecting with higher accuracy and corresponding to various mutations.

It is an object of the present invention to provide a kit for diagnosing infectious diseases caused by Severe Fever with Thrombocytopenia Syndrome (SFTS) virus.

Another object of the present invention is to provide a method for diagnosing the cause of a severe febrile thrombocytopenia syndrome (SFTS) viral disease.

Another object of the present invention is to provide a mutant gene for a diagnostic marker of severe recessive thrombocytopenia syndrome (SFTS) viral disease.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for a thorough understanding of the present invention, various specific details, such as specific forms, compositions and processes, etc. are set forth. However, certain embodiments may be practiced with one or more of these specific details, or other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific details in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the context of “one embodiment” or “implementation” expressed in various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, shapes, compositions, or properties may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined in the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

Recombinase polymerase amplification (RPA) is a method for rapid isothermal amplification of nucleic acids. Amplification depends on the specific combination of enzymes and proteins (recombinant enzyme, single-stranded binding protein and strand displacing DNA polymerase) used at a constant temperature. Real-time detection of RPA amplification products is possible through the exo-probe. Fluorescent substances are fluorophores and quenching substances through exonuclease III cleavage in the internal free base mimic of the hybridized exo-probe (such as tetrahydrofuran (THF)). (quencher) or the like can be used. The fluorescence signal is measured in real time with a 1.2 kg point-of-care scanner including a laptop (ESEQuant Tubescanner device, Qiagen Lake Constance GmbH, Stockach, Germany). For example, reverse primers labeled with biotin or horseradish peroxidase were used to investigate the use of different labeling strategies to reduce the number of steps required for the assay. This is a method to optimize the amplification temperature used and to use a surface blocker. The combination of these changes is a way ^{to lower the limit of detection (LOD) of 1 * 10 -15} M to facilitate a much faster amplification and detection protocol (J. Clin. Microbiol., 50 (2012), pp. 2234-2238 ).

Reverse transcription-recombinase polymerase amplification (RT-RPA) is the addition of reverse transcriptase in the RPA method described above.

In the present specification, "primer" is an oligonucleotide which is a short-sequence nucleotide, which is specifically attached to the complementary position of the opposite strand of the target DNA or RNA to initiate amplification of the gene. As an oligonucleotide serving as an oligonucleotide, preferably, it may be an oligonucleotide that specifically binds to the Segment M gene of a severe recessive thrombocytopenia syndrome (SFTS) virus, more preferably the base of SEQ ID NO: 1 and/or SEQ ID NO: 2 As an oligonucleotide comprising a sequence, the oligonucleotide may be a sequence similar to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2 by 80% or more, 90% or more, 95% or more, or 99% or more.

In the present specification, the term "probe" is a broad concept including a probe specifically attached to the gene of interest to identify and/or detect the gene of interest, preferably severe recessive thrombocytopenia syndrome (SFTS) It may be a compound, antibody, protein, oligonucleotide, etc. that specifically binds to the Segment M gene of the virus, more preferably between the 27th base and the 28th base from 5'of the sequence represented by SEQ ID NO: 7 FAM is inserted into, THF is inserted between the 28th and the 29th base, and the BHQ is inserted between the 29th and 30th bases, or 30 from 5'of the sequence represented by SEQ ID NO: 8 As an oligonucleotide comprising a nucleotide sequence in which FAM is inserted between bases 31 and 31, THF is inserted between bases 31 and 32, and BHQ is inserted between bases 32 and 33, The oligonucleotide may be a sequence that is 80% or more, 90% or more, 95% or more, or 99% or more similar to the nucleotide sequence of the sequence. In addition, the probe may include one or more labels for easy detection.

In the present specification, "kit" refers to a screening device capable of predicting the presence of SFTS virus infection by amplifying and/or detecting RNA of severe febrile thrombocytopenia syndrome (SFTS) virus. There is no limitation as long as it is a form capable of confirming the presence of SFTS virus infection from a biological sample isolated from a suspected subject. Preferably, it may include a probe or primer set having a sequence complementary to the 3D gene of the SFTS virus, and more preferably may include the primer set of the present invention, or specifically bind to the SFTS virus Segment M. It may be a form including an antibody or an aptamer, but is not limited thereto as long as it is a form capable of predicting the presence of SFTS virus infection by amplifying and/or detecting the RNA of the SFTS virus.

In the present specification, "detection" relates to a method for determining whether a subject suspected of being infected with a severe febrile thrombocytopenia syndrome (SFTS) virus is infected with a virus, and by identifying the protein or RNA of the virus It means a way to check for virus infection.

In the present specification, the term "biological sample" refers to any sample containing the protein or RNA of the virus in a subject infected with the severe febrile thrombocytopenia syndrome (SFTS) virus, preferably blood, plasma , Serum, bone marrow, tissue, cells, saliva, sputum, fur, urine, and the like, but are not limited thereto as long as it is a sample capable of confirming the protein or RNA of the virus.

In order to achieve the above object, the present invention provides a kit for diagnosing severe recessive thrombocytopenia syndrome comprising a primer base pair or probe corresponding to SEQ ID NO: 3 or SEQ ID NO: 6.

In one embodiment of the present invention, the primer base pair is SEQ ID NO: 1 and 2 or SEQ ID NO: 4 and 5. In another embodiment of the present invention, in the probe, FAM is inserted between bases 27 and 28 from 5'of the sequence represented by SEQ ID NO: 7, and THF is inserted between bases 28 and 29, , A base sequence in which BHQ is inserted between the 29th base and the 30th base, or FAM is inserted between the 30th base and the 31st base from 5'of the sequence represented by SEQ ID NO: 8, and the 31st base and the 32nd base THF is inserted between bases, and BHQ is inserted between the 32nd base and the 33rd base. In another embodiment of the present invention, the severe febrile thrombocytopenia syndrome is caused by the SFTS virus. In another embodiment of the present invention, the primer base pair is to detect the SFTS virus in the specimen. In another embodiment of the present invention, the specimen is one of whole blood, plasma, serum, or small small mite to be diagnosed.

In order to achieve the above object, the present invention provides a method for detecting severe fever thrombocytopenia syndrome virus in in-vitro , comprising the step of adding a probe or primer base pair corresponding to SEQ ID NO: 3 or SEQ ID NO: 6 to a sample. do.

In one embodiment of the present invention, in the probe, FAM is inserted between the 27th base and the 28th base from 5'of the sequence represented by SEQ ID NO: 7, and THF is inserted between the 28th and the base and 29th base. , A base sequence in which BHQ is inserted between the 29th base and the 30th base, or FAM is inserted between the 30th base and the 31st base from 5'of the sequence represented by SEQ ID NO: 8, and the 31st base and the 32nd base THF is inserted between bases, and BHQ is inserted between the 32nd base and the 33rd base. In another embodiment of the present invention, the primer base pair is SEQ ID NO: 1 and 2 or SEQ ID NO: 4 and 5. In another embodiment of the present invention, it includes the step of reverse transcription-recombinase polymerase amplification (RT-RPA) with the sample. In another embodiment of the present invention, the reaction temperature in the RT-RPA step is 37 ℃ to 42 ℃. In another embodiment of the present invention, the reaction time in the RT-RPA step is 5 minutes to 20 minutes.

In order to achieve the above object, the present invention provides an immunogenic composition for severe febrile thrombocytopenia syndrome (SFTS) virus, including the virus according to SEQ ID NO: 3 or SEQ ID NO: 6.

In one embodiment of the present invention, the composition further comprises an adjuvant.

The severe febrile thrombocytopenia syndrome diagnostic kit provided by the present invention has an effect of effectively and rapidly detecting and diagnosing SFTS virus infection in an individual, thereby improving the prevention and treatment effect against SFTS virus infection.

1 is a graph evaluating the efficiency of primers and probes for SFTS virus.
2 is an image of performing RT-RPA reaction to detect SFTS virus. 2A is a RT-PRA product loaded with 5 μl, B is a RT-PRA product loaded with 2.5 μl, C is a RT-PRA product loaded with 1.0 μl, and D is a reagent composition for RT-RPA reaction. Show.
3 is a diagram showing an RT-RPA reaction for detecting SFTS virus using a basic RT kit with different concentrations of primers. 3A shows 1.0 μl loading of RT-PRA product, B shows 5.0 μl loading of RT-PRA product, C shows the composition of each primer added to 10 pM, and D shows the composition of each primer added to 7 pM. The composition is indicated, and E indicates the composition of each primer added with 3 pM. In the gel image, even numbered lanes are 10 pg of SFTS DNA added to the reaction mixture, and odd numbered lanes are only distilled water.
4 is a diagram showing a comparison of RT-RPA reactions for detecting SFTS virus using a basic RT kit according to reaction time. 4A shows the amplification product generated by the RPA reaction was confirmed by agarose gel electrophoresis. 4B shows the components of the RT-RPA reaction. 1.0 μl of RT-PRA product was loaded into each lane.
5 is a diagram showing a comparison of RT-RPA reactions for detecting SFTS virus using a basic RT kit according to the reaction temperature. Fig. 5A shows that the agarose gel electrophoresis was performed for 20 minutes at a temperature range of 5°C to 55°C, respectively. Fig. 5B shows the implementation at a temperature range of 37°C to 42°C, respectively. 1.0 μl of RT-PRA product was loaded into each lane.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

Example 1: SFTS virus detection primer and probe set

The primer sets and probes for SFTS virus detection are summarized below.

-Forward primer (SEQ ID NO: 1) (also referred to herein as 1891 F):

5'-ATCAACCTAAAATGTAAGAAGTCATCATCATATTT-3'

-Reverse primer (SEQ ID NO: 2) (also referred to herein as 2194 R):

5'-CACATGGAGATACTTTCCAGATGATCCCATGTG-3'

-Probe (SEQ ID NO: 7) (also referred to herein as 38 P):

5'-GCCCCCCTCATTTCACATCCAACTCCTTCGATGATTGGGCAGGT-3'. However, FAM is inserted between the 27th base and the 28th base from 5'of the sequence, THF is inserted between the 28th and the 29th base, and BHQ is inserted between the 29th and 30th bases .

Example 2: SFTS virus detection primer and probe set

The primer sets and probes for SFTS virus detection are summarized below.

-Forward primer (SEQ ID NO: 4) (also referred to herein as 1 F):

5'-TCGATGAGATGGTCCATGCTGATTCTAAACTTGTT-3'

-Reverse primer (SEQ ID NO: 5) (also referred to herein as 197 R):

5'-TGCATTTCAGGTTAATCCTCTTAACTTTGATTTTG-3'

-Probe (SEQ ID NO: 8) (also referred to herein as 38 P):

5'-TGTAAGCAAGGGAGCGGAAATATGAAGGAAGGCACAACTGGGAGGGC-3'. However, FAM is inserted between the 30th base and the 31st base from 5'of the sequence, THF is inserted between the 31st and the base and 32nd base, and BHQ is inserted between the 32nd and 33rd bases .

The present inventors devised an effective diagnostic kit comprising a sequence capable of detecting SFTS with higher accuracy and responding to various mutations (Example 1 is SEQ ID NO: 3 and Example 2 is SEQ ID NO: 6) and sequences thereof I did. The gene of SEQ ID NO: 3 or SEQ ID NO: 6 was serially diluted from 0.01 pg to 10 ng. The primers and probes were mixed in an EXO RT kit (TwistDx Co. UK) to perform reverse transcription-recombinase polymerase amplification (RT-RPA). However, it is not limited to the above kit. This RT-RPA reaction was carried out on a T8 UV scanner at 39°C for 20 minutes. The RT-RPA reaction well amplified the SFTS membrane glycoprotein gene diluted to 0.01 pg. All samples diluted from 0.01 pg to 10 ng showed a positive reaction within 10 minutes (Fig. 1).

SEQ ID NO: 3: ctcaaaatta aggttaagag gatcaaccta aaatgtaaga agtcatcatc atattttgtt cctgatgccc ggtccaggtg tacatcagtg aggagatgtc gttgggcagg agactgccag tctgggtgcc cccctcattt cacatccaac tccttttctg atgattgggc aggtaagatg gacagggctg gtctaggatt cagtggctgc tctgatggat gtggaggagc agcctgcggc tgctttaatg cggccccttc atgcatcttc tggaggaaat gggtagagaa tccacatggg atcatctgga aagtatctcc atgtgccgca tgggtcccat cagcagtcat

SEQ ID NO: 6: gatgagatgg tccatgctga ttctaaactt gtttcgtgta agcaagggag cggaaatatg aaggaatgtg tcacaactgg gagggcgctc cttcctgcag tgaacccagg acaagaggca tgtctgcact tcacggcaca tggattagcccg gactcaaaatcccg gactcaaaa

Example 3: RT-RPA reaction mixture composition and reaction conditions

The RT-RPA reaction to detect SFTS virus was performed using the basic RT kit. SFTS was serially diluted from 0.0001 ng to 10 ng. As shown in Fig. 2, even if only 1 µl of the 50 µl reaction solution was loaded, it was sufficiently observed on the gel with the naked eye. From this, it was found that sufficient RPA amplification occurred within 20 minutes. The amplification product (amplicon) generated by the RT-RPA reaction was confirmed by agarose gel electrophoresis (FIG. 2).

In addition, RT-RPA reaction for detecting SFTS virus was performed using a basic RT kit with different concentrations of primers. The amplification product generated by the RT-RPA reaction was confirmed by agarose gel electrophoresis (FIG. 3). Each of the RT-PRA products was individually loaded with 1.0 μl (see FIG. 3A) and 5.0 μl (see FIG. 3B). Each primer was changed to 10pM, 7pM, and 3pM and added (see C to E in FIG. 3). As shown in FIG. 3, even numbered lanes in the gel image indicate that 10 pg of SFTS DNA was added to the reaction mixture, and odd numbered lanes indicated that only distilled water was added.

In addition, the RT-RPA reaction for detecting the SFTS virus was compared using the basic RT kit according to the reaction time. As shown in Fig. 4B, the components of the RT-RPA reaction were prepared. 1.0 μl of RT-PRA product was loaded into each lane. The amplification product generated by the RPA reaction was confirmed by agarose gel electrophoresis (see A in FIG. 4). The RT-RPA reaction using the basic RT was able to observe the amplified product on the gel from 5 minutes later. In particular, the darkest band could be confirmed in the sample reacted for up to 20 minutes.

In addition, RT-RPA reactions for detecting SFTS virus were compared using the basic RT kit according to the reaction temperature. As shown in C of FIG. 5, the composition was prepared as the components of the RT-RPA reaction. 1.0 μl of the RT-PRA product was loaded into each lane, and each was carried out for 20 minutes at a temperature range of 5° C. to 55° C. (FIG. 5A). Bands were found in the lanes at 35°C and 45°C. Thereafter, 1.0 μl of the RT-PRA product was loaded into each lane, and each was carried out again at a temperature range of 37°C to 42°C for 20 minutes (FIG. 5B). As a result, RT-RPA reaction did not react at 5, 15, 25, and 55°C. However, from 37°C to 42°C, there was no significant difference in the RT-RPA reaction, and it was confirmed that all of them reacted well.

In conclusion, the method according to the present application has the advantage of being applicable in a laboratory where a real-time PCR device, a T8 UV scanner, or a T16 scanner cannot be used. It was expected that a gene sample of up to 0.1 pg could be detected by reacting at a temperature of 37°C to 42°C, preferably at 39°C for 5 minutes or more, preferably 5 minutes to 20 minutes.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Scorpiogen Co. <120> KIT FOR DIAGNOSING INFECTION DUE TO SEVERE FEVER WITH THROMBOCYTOPENIA SYNDROME VIRUS <130> PDPB187214 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 atcaacctaa aatgtaagaa gtcatcatca tattt 35 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 cacatggaga tactttccag atgatcccat gtg 33 <210> 3 <211> 350 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 3 ctcaaaatta aggttaagag gatcaaccta aaatgtaaga agtcatcatc atattttgtt 60 cctgatgccc ggtccaggtg tacatcagtg aggagatgtc gttgggcagg agactgccag 120 tctgggtgcc cccctcattt cacatccaac tccttttctg atgattgggc aggtaagatg 180 gacagggctg gtctaggatt cagtggctgc tctgatggat gtggaggagc agcctgcggc 240 tgctttaatg cggccccttc atgcatcttc tggaggaaat gggtagagaa tccacatggg 300 atcatctgga aagtatctcc atgtgccgca tgggtcccat cagcagtcat 350 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 4 tcgatgagat ggtccatgct gattctaaac ttgtt 35 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 5 tgcatttcag gttaatcctc ttaactttga ttttg 35 <210> 6 <211> 197 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 6 gatgagatgg tccatgctga ttctaaactt gtttcgtgta agcaagggag cggaaatatg 60 aaggaatgtg tcacaactgg gagggcgctc cttcctgcag tgaacccagg acaagaggca 120 tgtctgcact tcacggcaca tgggagcccg gactcaaaat gtcacaaaat caaagttaag 180 aggattaacc tgaaatg 197 <210> 7 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 7 gcccccctca tttcacatcc aactccttcg atgattgggc aggt 44 <210> 8 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 8 tgtaagcaag ggagcggaaa tatgaaggaa ggcacaactg ggagggc 47

Claims (14)

A kit for diagnosing severe recessive thrombocytopenia syndrome comprising a primer base pair or probe corresponding to SEQ ID NO: 3 or SEQ ID NO: 6,
The primer base pair is a primer base pair represented by SEQ ID NOs: 1 and 2; Or a primer base pair represented by SEQ ID NOs: 4 and 5,
The probe is a probe represented by 5'-GCCCCCCTCATTTCACATCCAACTCCT [FAM-dT] T [THF] C [BHQ-dT] GATGATTGGGCAGGT-[3-block]; Or 5'-TGTAAGCAAGGGAGCGGAAATATGAAGGAA [FAM-dT] G [THF] G [BHQ-dT] C ACAACTGGGAGGGC-[3'-block],
The diagnostic kit is a reverse transcription-recombinase polymerase amplification (RT-RPA) Yongin, diagnostic kit. delete delete The method of claim 1,
The severe febrile thrombocytopenia syndrome is caused by the SFTS virus, a diagnostic kit. The method of claim 1,
The primer base pair is to detect the SFTS virus in the specimen, diagnostic kit. The method of claim 5,
The test kit is one of whole blood, plasma, serum, or small small ticks to be diagnosed. A method for detecting a severe recessive thrombocytopenia syndrome virus in in-vitro , comprising the step of adding a probe or primer base pair corresponding to SEQ ID NO: 3 or SEQ ID NO: 6 to the sample,
The primer base pair is a primer base pair represented by SEQ ID NOs: 1 and 2; Or a primer base pair represented by SEQ ID NOs: 4 and 5,
The probe is a probe represented by 5'-GCCCCCCTCATTTCACATCCAACTCCT [FAM-dT] T [THF] C [BHQ-dT] GATGATTGGGCAGGT-[3-block]; Or 5'-TGTAAGCAAGGGAGCGGAAATATGAAGGAA [FAM-dT] G [THF] G [BHQ-dT] C ACAACTGGGAGGGC-[3'-block],
A method for detecting, comprising the step of performing reverse transcription-recombinase polymerase amplification (RT-RPA) with the sample. delete delete delete The method of claim 7,
The reaction temperature in the RT-RPA step is 37 ℃ to 42 ℃, detection method. The method of claim 7,
The reaction time in the RT-RPA step is 5 minutes to 20 minutes, detection method. delete delete

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