JPH0819400A - Detection of virus involved in hemorrhagic fever with renal syndrome - Google Patents

Abstract

PURPOSE:To quickly detect in high sensitivity virus involved in hemorrhagic fever with renal syndrome (HFRSV) by conducting an amplification through reverse transcription and the following PCR by using an nucleic acid with a base sequence specific to HFRSV and another nucleic acid with the complementary chain sequence. CONSTITUTION:A nucleic acid is separated from a specimen and then subjected to reverse transcription with a primer such as nucleic acid fragment(s) of formula I and/or formula II, and the resultant transcripted DNA is subjected to 1st stage PCR with said nucleic acid fragment(s) and amplified, and then further subjected to 2nd stage PCR with nucleic acid fragments of formula III and Formula IV to effect further amplification into a nucleic acid of e.g. formula V and a second nucleic acid with the complementary chain sequence. By amplifying part of these nucleic acids, HFRSV effective for e.g. diagnosing hemorrhagic fever with renal syndrome (HFRS) is quickly detected in high sensitivity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting renal symptomatic hemorrhagic fever virus, and more particularly to a method for genetically detecting a specific nucleic acid region of the virus. The present invention also relates to a detection primer used for such genetic engineering detection.

[0002]

[Prior Art] renal symptomatic hemorrhagic fever (hemorrhagic fever wi
th renal syndrome (hereinafter, abbreviated as HFRS) is one of the zoonotic diseases and is transmitted by rats. In humans, it causes acute infection with renal dysfunction as the main symptom, and persistent infection is often found in rats (field mice or rats in the laboratory) and is the source of infection. It is widely distributed from Far East Asia to Russia and Europe, and recently an outbreak of patients has been reported in the United States [Science (Scienc
e), Vol. 262, pp. 914-917 (1993)].
The annual number of patients has reached hundreds of thousands in the world, and in Japan, cases of infection and death from laboratory rats have been reported. HFRS virus (hereinafter abbreviated as HFRSV)
Since the isolation of the Hantaan strain from the lungs of the Korean bursian rat in 1978 by Lee et al.
More than one species of HFRSV has been isolated from lung, blood, tumor, pancreas, etc., and B-1 strain has also been isolated from Japanese experimental animal rat. The most pathogenic in the Far East Asia region is the Hantan strain HFRSV, which has a mortality rate of 10 to 10.
Up to 15% [Anitex, Volume 5, Issue 6, 27]
3 to 276 (1993)]. B-1 strain is Seoul HF
It is a type of virus that is currently prevalent in Japan, using RSV in rats as a host, and has a weaker pathogenicity than the Hantan strain, but has a high infectivity. HFRSV is a bunyavirus (buny
This is a single-stranded RNA virus belonging to the family avirus), and its RNA is divided into large, medium, and small (L chain, M chain, S chain) trisegments. HFRSV RNA replication is RNA-dependent R
In addition to the (-)-strand genomic RNA (vRNA) in the infected cells in which the virus is active, it is complementary to the vRNA required for vRNA replication (+).
There are strand RNA (cRNA) and (+) strand mRNA. The diagnosis of HFRS depends on clinical symptoms,
Currently, serodiagnosis is performed. Recently, Polymerase Chain Reaction
The diagnosis is also performed by the method (ction, PCR) (Japanese Patent Laid-Open No. 3-120,000, Science, supra).

[0003]

Serodiagnosis is performed by a detection method such as an indirect fluorescent antibody method. The antibody detection method has a high specificity, which enables early diagnosis after the onset of HFRS. However, the antibody detection method indirectly indicates the state of the virus, and does not directly indicate the presence of the virus. HFRSV particles are composed of four types of proteins and genes, and the characteristics of HFRSV are defined by the genes. If the nucleic acid sequence characteristic of HFRSV is amplified by the PCR method, the presence of the virus can be determined directly, with high sensitivity, quickly and earlier. However, the PCR methods reported so far have been insufficient in terms of sensitivity to detect clinically isolated specimens, particularly viruses that are present only in trace amounts such as in blood. The object of the present invention is to provide PC in the HFRSV nucleic acid region.
It is to clarify a region that can be amplified with higher sensitivity by the R method, and to provide a detection method therefor and a HFRSV detection primer used therefor.

[0004]

SUMMARY OF THE INVENTION The present invention will be outlined. The first invention of the present invention relates to a method for detecting HFRSV, which comprises HF
In the method for detecting RSV, a part of at least one nucleic acid selected from the group consisting of the nucleic acids represented by SEQ ID NOs: 1 to 5 in the sequence listing and their complementary strand sequences is obtained by reverse transcription and subsequent two-step PCR. It is characterized by including a step of amplifying. The second invention of the present invention relates to a method for detecting HFRSV including the following steps. (A) a step of separating the nucleic acid from the sample; (b) the separated nucleic acid is represented by the nucleic acid fragment represented by SEQ ID NO: 6 in the sequence listing or its mutant sequence, and / or the SEQ ID NO: 7 in the sequence listing. Reverse transcribing with a primer selected from the nucleic acid fragment or its mutated sequence to obtain reverse transcribed DNA, (c) the reverse transcribed DNA is SEQ ID NO: 6 in the sequence listing.
A step of carrying out a first-step PCR to amplify DNA by a combination of the nucleic acid fragment represented by or the mutant sequence thereof and the nucleic acid fragment represented by SEQ ID NO: 7 of the sequence listing or the mutant sequence thereof, and (d) amplified DNA A step of carrying out a second-stage PCR with a combination of the nucleic acid fragment represented by SEQ ID NO: 8 in the sequence listing or its mutant sequence and the nucleic acid fragment represented by SEQ ID NO: 9 in the sequence listing or its mutant sequence to amplify DNA ,
And (e) a step of detecting the amplified DNA. Furthermore, a third invention of the present invention is the HFRS comprising the nucleic acid fragments represented by SEQ ID NOs: 6 to 9 in the sequence listing, or a mutant sequence thereof.
It relates to a V detection primer.

Diagnosis of HFRS can be made by detecting the HFRSV gene. There are various methods for detecting genes, but in order to realize highly sensitive detection, two-step P
CR is most preferred. Therefore, the primers may be designed in consideration of the gene sequences of HFRSV of Hantan genus, which is the most pathogenic in Far East Asia, and Seoul, which is widely distributed in Japan. The inventors of the present invention are H of Hantan and Seoul.
Two-step PC with high homology in FRSV gene sequence
As a result of examining various primers targeting these genes in order to determine a region that can be detected with R with high sensitivity, a target nucleic acid region was found in the M chain G ₂ region, and the present invention was completed.

The present invention will be specifically described below step by step. The Hantan genus HFRSV includes, for example, Hantan genus 76-118 and Lee strains, and the Seoul HFRSV includes, for example, Seoul genus B-1 strain, 80.
-39 strain and SR-11 strain are known. The cDNAs of the genomic RNAs of these strains have already been cloned, and their nucleotide sequences are shown in Virology, No. 157.
Volume, 31-39 (1987), Journal of
General Birology (J. Gen. Virol.), 69th
Vol., Pp. 1949-1955 (1988), Nucleic Acids Research,
Volume 18, 4936-4937 (1990), Virus Research, Volume 19, 47
Pp. 58 (1991), virology, volume 176, volume 1.
14-125 (1990), respectively.

From these base sequences, HFR HFR
First-stage PCR using a primer for reverse transcription of the selected nucleic acid region and a nucleic acid region having high homology to SV and Seoul HFRSV as a template (hereinafter 1
A primer pair for amplification by stPCR) and a primer pair for amplification by second-stage PCR (hereinafter abbreviated as 2nd PCR) using the DNA amplified by 1st PCR as a template are designed and synthesized. The RNA region common to the genus Hantan and the genus Seoul selected by the present inventors includes base numbers 791 to 1224 (A region) of the M chain (cDNA) of the Seoul genus B-1 strain shown in SEQ ID NO: 10. ), Base numbers 2079 to 2522 (B region), base numbers 2068 to 2585 (C region: two-step PCR amplification region described in the above science), base numbers 2495 to 28.
There are regions corresponding to the 77th (D region) and the base numbers 3083 to 3288 (E region), respectively, and these are nucleic acid regions having high homology between Hantan genus and Seoul genus HFRSV.

Next, a primer for reverse transcription of these nucleic acid regions, a primer pair for 1st PCR, 2nd
Make primer pairs for PCR. As a primer for reverse transcription, any one of the primer pairs for 1st PCR may be used, but if they are simultaneously used as a primer pair, vRNA of the (−) strand and cRN of the (+) strand in the sample are used.
A and mRNA can be reverse transcribed at the same time, which is suitable as a highly sensitive detection method.

Any primer can be used as long as it can anneal to the above-mentioned selected region and carry out the desired amplification.
A primer pair for reverse transcription and 1st PCR (hereinafter abbreviated as primer pair A) prepared by the present inventors, and 2ndP
Table 1 shows one example of a primer pair for CR (hereinafter abbreviated as primer pair B).

[0010]

[Table 1] Table 1 ──────────────────────────────────── Nucleic acid region Primer pair A Primer pair B Sequence Listing Sequence Listing Primer Name Sequence Number Primer Name Sequence Number ───────────────────────────────────── Area A HS-HF1-1 11 HS-HF1-2 13 HS-RS1-1 12 HS-RS1-2 14 ───────────────────────── ──────────── Area B HS-HF2-1 15 HS-HF2-2 17 HS-RS2-1 16 HS-RS2-2 18 ────────────── ──────────────────────── Region C HS-HF4-1 19 HS-HF4-2 21 HS-RS4-1 20 HS-R 4-2 22 ──────────────────────────────────── D area HS-HF5-1 23 HS- HF5-225 HS-RS5-1 24 HS-RS5-2-2 26 ─────────────────────────────────── ── E region HS-HF3-1 6 HS-HF3-2 8 HS-RS3-1 7 HS-RS3-2 9 ─────────────────────── ──────────────

The HFRSV RNA to be detected using the primer pair A and the primer pair B is, for example, A54.
9 (ATCC CCL185), Vero (ATCC
CCL81) or Vero-E6 (Osaka University Research Institute for Microbial Diseases) cells infected with Hantan strain or Seoul strain, and organs such as lung, spleen, and brain of HFRSV-infected animals, blood, saliva, Urine, feces, and even HFR
It can be prepared from body fluids such as blood, saliva, and urine of S patients.

[0012] Regarding the PCR method, RNA containing Taq DNA polymerase and reverse transcriptase
-A PCR kit is commercially available from Takara Shuzo, and an automatic gene amplification device (Perkin Elmer Co.) is also commercially available from Takara Shuzo.

Using these and the primer pairs shown in Table 1, H
FRSV is detected. In the reverse transcription reaction, for example, an AMV-derived reverse transcriptase and a primer pair A are used, and four types of substrates (dATP, dCTP, dGTP, d) are used.
TTP) is added and heated (42 ° C.) to generate template R
Single-stranded cDNA is synthesized from NA. The subsequent two-step PCR is, for example, a thermostable tack DNA polymerase and a primer pair A
The target gene is amplified by repeating the temperature cycle consisting of the step of denaturing A (94 ° C.), the step of annealing primer DNA (50 ° C.) and the step of enzymatically synthesizing a complementary DNA strand (72 ° C.) any number of times. Next, 2nd PCR is performed using the gene as a template. As the 2nd PCR, for example, a step of denaturing DNA (94 ° C.) using a thermostable tack DNA polymerase and primer pair B, primer DNA
The target gene is amplified by repeating the temperature cycle consisting of the step of annealing (45 ° C.) and the step of enzymatically synthesizing a complementary DNA strand (72 ° C.) any number of times.

Vero-E infected with Seoul B-1 strain
Table 2 shows the detection limits when each nucleic acid region was detected using 6 cell-derived RNA (0.001 to 0.1 pg) as a template and using primer pair A and primer pair B shown in Table 1.

[0015]

[Table 2] Table 2 ─────────────────────────── Nuclear acid region Detection limit limit ────────── ────────────────── A region 0.1 pg B region 0.1 pg C region 0.1 pg D region 0.1 pg E region 0 .01 pg ────────────────────────────

As shown in Table 2, only the E region can be detected with a sensitivity 10 times that of the other regions, and this region is the most excellent site for detecting HFRSV.

Next, blood was collected from HFRSV-infected rats,
An example of detecting HFRSV in blood is shown below. That is, blood was collected from a rat infected with Seoul B-1 strain,
1 ml of blood treated with 3.1% sodium citrate was diluted 2-fold with phosphate buffer, and then Ficoll (specific gravity: 1.0
940) Layer on 1 ml and centrifuge at 25 ° C. to collect lymphocytes. After washing the lymphocytes thoroughly with phosphate buffer, guanidine isothiocyanate is added to the lymphocytes to lyse the cells. After the lysed cell extract is treated with phenol and chloroform, 2-propanol precipitation is performed to recover RNA. Table 3 shows the results of amplification of the E region using this RNA as a template and the primer pair A and the primer pair B corresponding to the E region shown in Table 1. As a control, rat blood cells before infection were used and treated in the same manner.

[0018]

[Table 3] Table 3 ────────────────────────── Presence of infection Presence of DNA amplification ─────────── ──────────────── Yes Yes No No ───────────────────────────

As shown in Table 3, amplification of specific DNA was observed only in the blood of infected rats, and the HFRSV-derived RNA in a blood sample can be simply and highly sensitively detected by detecting the E region. You can

As described above, the nucleic acid region useful for HFRSV detection is determined by the present invention. This nucleic acid region is Seoul B
-1 strain is the region represented by SEQ ID NO: 1 in the sequence listing,
The Seoul genus 80-39 strain is the region represented by SEQ ID NO: 2 in the sequence listing, and the Seoul genus SR-11 strain is the region represented by SEQ ID NO: 3 in the sequence listing. Also Hantan 76-1
In 18 strains, it is the region represented by SEQ ID NO: 4 in the sequence listing,
In the Hantan genus Lee strain, it is a region represented by SEQ ID NO: 5 in the sequence listing. By amplifying these regions or a part of their complementary strand sequences by reverse transcription and two-step PCR, HFRSV can be highly sensitive. Can be detected. Hereinafter, the regions represented by SEQ ID NOS: 1 to 5 in the sequence listing will be referred to as E'regions.

One example of the primer pair A and the primer pair B for detecting the E'region and its complementary strand sequence is SEQ ID NOs: 6 and 7 and SEQ ID NOs: 8 and 9 of the Sequence Listing, respectively.
The nucleic acid fragment shown in. The primer used in the present invention may be a mutated sequence of these nucleic acid fragments, and the mutated sequence may be any as long as it functions in the detection of the E'region and its complementary strand sequence. A part of the base sequence of the basic primer represented by Nos. 6 to 9 is deleted, or is substituted or added with another base, and more specifically, Hantan genus and Seoul genus HFRSV. The nucleotide sequence corresponding to the corresponding portion of the same gene in the mutant strain of. It should be noted that there is preferably no mutation near the 3'end of the primer, which is thought to greatly affect the efficiency of the primer extension reaction, or even if there is any mutation, it is more preferable that it is 5 '. Make sure there are mutations near the ends. Further, since it corresponds to the mutation in the E'region and its complementary strand sequence, it is also a good method to replace the base in the primer corresponding to the mutation part with inosine. The nucleic acid fragments represented by SEQ ID NOS: 27 to 30 in the sequence listing are examples of the respective mutated sequences of the basic primers represented by SEQ ID NOS: 6 to 9 in the sequence listing. These mutant sequences can be used to efficiently amplify the E'region and its complementary strand sequence. Further, the mutant sequence and the basic primer may be used in combination.

Next, the amplified DNA can be easily detected by binding a detectable label to the primer of the present invention. Here, the label may be either a non-radioactive substance or a radioactive substance, but is preferably a non-radioactive substance. As the non-radioactive label, a fluorescent substance that can be directly measured (for example, fluorescein and its derivative (fluorescein isothiocyanate, etc.), rhodamine and its derivative (tetramethylrhodamine isothiocyanate, Texas red, etc.)), Examples include chemiluminescent substances (for example, acridine) and substances that emit delayed fluorescence (DTTA: manufactured by Pharmacia).

Hereinafter, HFR using the primer of the present invention
The SV detection method will be described in detail. (1) Specimen: As a specimen for detecting the presence or absence of the target HFRSV, animal cells infected with HFRSV described above, organs obtained from infected animals, blood, etc., blood obtained from patients, saliva, urine Etc. If blood is used among these, continuous diagnosis of infected animals and patients can be easily performed. RNA for detecting HFRSV in blood
Although there are various methods for preparing RNA from blood, for example, lymphocytes may be separated by the Ficoll method and RNA may be prepared from the lymphocytes.
A method of preparing RNA from a small amount of blood using a cationic surfactant [Nature, No. 36]
2, pp. 186-188 (1993)], and at this time, Catrimox-1 (Catrimox-1).
4: manufactured by Biotechnology Co., Ltd.) is preferable.

(2) Reverse transcription and gene amplification: By adding the primer pair A of the present invention to the above sample, if the desired HFRSV is present in the sample, the E'region and its complementary chain region by the reverse transcriptase. Reverse transcription can be performed. Then, using primer pair A of the present invention, 1stP
By performing 2nd PCR using CR and the primer pair B of the present invention, a part of the reverse transcribed E region can be efficiently amplified.

(3) Detection: The gene of interest generated by the two-step PCR was electrophoresed [Saiki, Science, 230, 1350-1354 (198).
5)], or dot hybridization using a labeled probe [Saiki, Nature, Vol. 324, pp. 163-166 (1986)] and the like. As the probe, the probe DN is used within the amplification region.
Design and manufacture A. The probe labeling method is not limited to the radioisotope labeling method, and any known method such as enzyme labeling, fluorescent labeling, biotin / avidin labeling, and chemiprobe labeling method may be used.

Primer pair A and primer pair B of the present invention
HFRSV can be easily detected by preparing the above as a kit. It is to be noted that the RN of blood HFRSV can be obtained by preparing reagents for collecting lymphocytes from blood, reverse transcriptase, PCR reagents, etc. in the kit.
A can be detected more quickly, conveniently and with high sensitivity. The reagent in the kit may be a solution or a lyophilized product.

[0027]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of Primer DNA Primer DNAs represented by SEQ ID NOS: 6 to 25 in the Sequence Listing were synthesized using a DNA synthesizer manufactured by Applied Biosystems, deprotected, and ion-exchanged. HPLC (T
SK gel, DEAE-2SW column)
Desalting was carried out using Sep-pak C ₁₈ (Waters) to obtain each DNA.

(2) Preparation of intracellular RNA infected with Seoul genus B-1 strain Animal cell Vero E6 was infected with Seoul genus B-1 strain (Osaka University Research Institute for Microbial Diseases) 50 ml of Eagle MEM medium using Roux bottle CO at the culture scale of
₂ Cultured in an incubator for 7 days. The cells were collected by centrifugation and guanidine isothiocyanate was added to lyse the cells. The lysed cell extract was treated with phenol and chloroform, followed by 2-propanol precipitation to recover RNA.

(3) Reverse transcription of primers prepared in various regions of M chain and comparison of amplification ability by two-step PCR Using 5 kinds of primer pair A and primer pair B shown in Table 1, detection of HFRSV The comparison was performed using an RNA-PCR kit (Takara Shuzo). The B-1 strain-infected intracellular RNA obtained in Example 1- (2) was diluted with Tris-EDTA buffer (pH 8.3) 10 ⁶ fold, 10 ⁷ fold, and 10 ⁸ fold, respectively, and 1 μl of a sample. Each primer pair A in Table 1, dNTP mixture in RNA-PCR kit,
Magnesium chloride, 10 times RNA-PCR buffer and water are added to bring the total volume to 18 μl, and the mixture is heated at 70 ° C. for 3 minutes and rapidly cooled. Reverse transcriptase and RNase inhibitor were added thereto to make a total volume of 20 μl, and after heating at 30 ° C. for 10 minutes, 4
The reaction was carried out at 2 ° C for 60 minutes. The reverse transcriptase was inactivated at 99 ° C. for 5 minutes and cooled, and then the tack DNA polymerase, 10
RNA-PCR buffer, magnesium chloride, primer pair A corresponding to each, and water were added to bring the total amount to 100 μm.
l. After adding 100 μl of mineral oil (Takara Shuzo Co., Ltd.) to the upper layer of this reaction solution, automatic cycler 480 (Thermal Cycler 480) automatic gene amplification device was added.
The amplification reaction was performed by (Perkin Elmer). The reaction conditions were 94 ° C., 30 seconds denaturation → 55 ° C., 30 seconds primer annealing → 72 ° C., 30 seconds synthesis reaction cycle, 40 cycles.

After the reaction, 3 μl (3%) of the reaction solution was transferred to another tube and treated with tack DNA polymerase and 10 times RN.
A-PCR buffer, magnesium chloride, dNTP mixture, the primer pair B shown in Table 1 corresponding to each, and water were added to make the total volume 100 μl. After adding 100 μl of mineral oil to the upper layer of this reaction solution, an amplification reaction was carried out by an automatic gene amplification device Thermal Cycler 480. The reaction conditions are denaturation at 94 ° C for 30 seconds →
Annealing of primer at 55 ° C. for 30 seconds → 72
40 cycles of 30 ° C. and 30 seconds of synthetic reaction were performed. After the reaction, after removing the upper layer mineral oil, take 10 μl of the reaction solution and take 3% NuSieve GTG Agarose-1% Sea-Kem Agarose (FM
(Company C) Gel electrophoresis and D with ethidium bromide
The NA was stained and the amplified DNA was confirmed.

As a result, when each of the primer pair A and the primer pair B shown in Table 1 was used, specific amplification was observed in the E region up to a 10 ⁷ -fold dilution, but in the A to D regions, 10 ⁶ Only amplification was observed in the fold dilution, and the E region was determined to be the optimum region for detecting HFRSV. At this time, 1 μl of a 10 ⁷ -fold diluted solution of RNA in cells infected with the B-1 strain
The amount of RNA therein was 0.01 pg.

(4) Preparation of kit AM primer for detecting E'region and its complementary strand sequence, AM
V-derived reverse transcriptase (Takara Shuzo), tack polymerase (Takara Shuzo), RNase inhibitor (Takara Shuzo), 10-fold RNA PCR buffer, substrate (dNTP mixture), magnesium chloride, and HFRSV shown in Table 4 with positive control RNA A detection kit was prepared. The reagent G in the kit is the same as in Example 1 above.
The RNA described in (2) was used.

[0034]

[Table 4] Table 4 ──────────────────────────────────── HFRSV detection kit (100 times) ─ ─────────────────────────────────── Reagent A ・ ・ ・ Reverse transcriptase 50 units / μl 10 μl Reagent B・ ・ ・ Tac DNA polymerase 5 units / μl 100 μl Reagent C ・ ・ ・ RNase inhibitor 40 units / μl 50 μl Reagent D ・ ・ ・ 10 times RNA-PCR buffer 2 ml ┌-100 mM Tris-HCl buffer, pH 8.3 └─ 500 mM potassium chloride reagent E ... 25 mM magnesium chloride 1.6 ml reagent F ... dNTP mixture 1.6 ml 2.5 mM each dATP, dCTP, dGTP, dTTP reagent G ... positive control RNA 10 pg / μl 50 μl Reagent H: Primer pair A 200 μl each HS-HF3-1 20 pmol / μl HS-RS3-1 20 pmol / μl Reagent I: Primer pair B 100 μl HS-HF3-2 20 pmol / μl HS-RS3-2 20 pmol / μl ────────────────────────────────────

Example 2 Detection of HFRSV in Blood of HFRSV-Infected Rats Blood samples were collected from rats infected with H-1 strain B-1 over time, and the collected blood was treated with 3.1% sodium citrate. After 2-fold dilution with acid buffer, it was layered with 1 ml of Ficoll (specific gravity: 1.0940) and centrifuged at 25 ° C. to collect the lymphocyte layer. After thorough washing with a phosphate buffer, guanidine isothiocyanate was added to the cells to lyse the cells. The lysed cell extract was treated with phenol and chloroform, followed by 2-propanol precipitation to recover RNA. The RNA was stored at -80 ° C and dissolved in 10 µl of Tris-EDTA buffer before use. Rats infected with B-1 strain of Hantan are B-
One strain (about 10 ³ ffu) was intraperitoneally administered to 6-week-old male rats to cause infection, and blood was collected 1 week and 2 weeks after infection (after the onset). Pre-infection rat blood was prepared as a control.

Using the RNA prepared from the above-mentioned animal blood and the kit described in Example 1- (4), HFRSV was detected as follows. RN prepared from the above-mentioned animal blood
1 μl of the solution of A and 1 part of the primer pair A in the kit described in Example 1- (4), dNTP mixture, magnesium chloride, 10 times RNA-PCR buffer, and water were added to make 18 μl, and the total amount was 70 ° C. Heated for 3 minutes and quenched. Reverse transcriptase and RNase inhibitor for one kit were added thereto to make the total volume 20 μl, and after heating at 30 ° C. for 10 minutes, reaction was performed at 42 ° C. for 60 minutes. After deactivating the reverse transcriptase at 99 ° C for 5 minutes and cooling, the tack DNA polymerase for 10 times of the kit, 10 times RNA-PCR buffer,
Magnesium chloride, primer pair A, and water were added to make the total volume 100 μl.

After adding 100 μl of mineral oil to the upper layer of this reaction solution, an amplification reaction was carried out by an automatic gene amplification device Thermal Cycler 480. The reaction conditions are 94
40 cycles of denaturation for 30 seconds at 50 ° C., annealing of primer for 30 seconds at 50 ° C., and synthesis reaction for 72 seconds at 72 ° C. were performed. After the reaction, 3 μl (3
%) To another tube and tack DN for one kit
A polymerase, 10 times RNA-PCR buffer, magnesium chloride, dNTP mixture, primer pair B,
Water was added to make the total volume 100 μl, 100 μl of mineral oil was added to the upper layer of this reaction solution, and then amplification reaction was carried out by an automatic gene amplification device Thermal Cycler 480. The reaction conditions are 94 ° C, denaturation for 30 seconds → 45 ° C,
Annealing of the primer for 30 seconds → 72 ° C., 40 seconds of a synthetic reaction cycle for 30 seconds. After the reaction, after removing the upper layer mineral oil,
A 1 μl aliquot was taken and subjected to 3% Nusieve GTG agarose-1% C-chem agarose gel electrophoresis, and the DNA was stained with ethidium bromide to confirm the amplified DNA.

As a result, a fragment (119 bp) of the size expected from the nucleotide sequence of the M chain of Seoul genus B-1 strain RNA was amplified only from the blood sample of infected rat.
As a result of analyzing the nucleotide sequence of the nucleic acid of the above DNA fragment by the dideoxy sequencing method, the genus Seoul
It was confirmed that the nucleotide sequence was derived from the -1 strain. HFRS
HFRSV in blood collected from V-infected rats over time
Table 5 shows the detection results of.

[0039]

[Table 5] Table 5 ─────────────────────────── 1 week before sensitization 2 weeks ─────────── ────────────────── ＋＋＋ ───────────────────────────

(In Table 5, − indicates no specific gene amplification, + indicates specific gene amplification, and ++ indicates strong amplification.) As shown in Table 5, the initial stage of infection The presence of the virus was confirmed by the method of the present invention even when it could not be detected by the conventional serological method (1 week after infection).

[0041]

According to the present invention, HFRSV is extracted from a sample,
It is possible to detect directly with high sensitivity in a short time,
Definitive diagnosis of renal symptomatic hemorrhagic fever has become easy to perform. The method for detecting HFRSV and the detection primer provided by the present invention are useful in fields such as diagnosis of HFRS patients, management of experimental animals, and environmental purification by monitoring HFRSV.

[0042]

[Sequence list]

SEQ ID NO: 1 Sequence length: 206 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic RNA Sequence: GGUGCACCAU CAUCAUAAAC UUUCUCGUUU UCUAACUCAG AGAUACCAUU UACCUUAUCC 60 AGAUGUGGUG CACUGGCUUG GAGGCCAGUU GAU ACAGCAUUUA 120 AAUGAAGAAC CACUAUGGCC ACCUUUCCCA GUAAUUUUGA CAGUAUUUUG UCCUCGUGAG 180 AGUGUUACAC UUUCUGCACC GUAACA 206

SEQ ID NO: 2 Sequence Length: 206 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Genomic RNA Sequence: GGUGCACCAU CAUCAUAAAC UUUCUCAUUU UCUAACUCAG AGAUUCCAUU UACCUUAUCU 60 AAAUGUGGUG CACUGGCUUG GAGACCAGUU AGA ACAGCACUUA 120 AAUGAGGAAC CACUAUGGCC ACCUUUCCCG GUAAUUUUGA CAGUAUUUUG UCCUCGUGAG 180 AGUGUCACAC UUUCUGCACC AUAACA 206

SEQ ID NO: 3 Sequence Length: 206 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Genomic RNA Sequence: GGUGCACCGU CAUCAUAAAC UUUCUCGUUU UCUAACUCAG AGAUACCAUU UACCUUAUCC 60 AGAUGUGGUG CACUGGCUUG GAGGACAUU GAUCCAUGUU ACAGCAUUUG 120 AAUGAAGAAC CACUAUGGCC ACCUUUCCCG GUAAUUUUGA CAGUAUUUUG UCCUCGUGAG 180 AGUGUCACAC UUUCUGCACC AUAACA 206

SEQ ID NO: 4 Sequence length: 206 Sequence type: Nucleic acid Number of strands: Single-stranded topology: Linear Sequence type: Genomic RNA Sequence: GGUGCCCCAU CAUCAUAUAC UUUACUAUUU UCUAUCUCAG AAAUCCCAUU UACCUUGUCA 60 AGGUGAGGUG CAGCAGCAUG GUGUCCAAUU GCAACACCUA 120 AAUGUUGAAC CACUAUGGCC ACCUUUCCCU GAUACCUUGA CUGUAUUUUG UCCUCUUGUC 180 AAUGUUACAC UCUCUGCACC AUAACA 206

SEQ ID NO: 5 Sequence length: 206 Sequence type: Nucleic acid Number of strands: Single-stranded topology: Linear Sequence type: Genomic RNA Sequence: GGUGCUCCAU CAUCAUAUUC UUUGCUAUUU UCCAUCUCAG AAAUCCCAUU GACCUUGUCU 60 AGGUGAGGUG CAGCAGCAUG GUGUCCAAUU GCAACACUUA 120 AAUGUUGAAC CACUAUGGCC ACCUUUUCCU GAUACCUUGA CUGUAUUUUG CCCUCUUGUU 180 AAUGUUACAC UCUCUGCACC AUAACA 206

SEQ ID NO: 6 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: TGTTATGGYG CAGAGAGTGT RAC 23

SEQ ID NO: 7 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GGTGCHCCRT CATCATAWWC TTT 23

SEQ ID NO: 8 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GGRAAAGGTG GCCATAGTGG TTC 23

SEQ ID NO: 9 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence: TCYAWCTCAG ARATHCCATT KAC 23

SEQ ID NO: 10 Sequence length: 3651 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to Genomic RNA Sequence: TAGTAGTAGA CTCCGCAAGA AACAGCAGTT AAATAACAGC AGGATCATGT GGAGTTTGCT 60 ATTACTGGCC GCTTTAGTTG GTCAAGGCTT TGCATTAAAA AATGTGTTTG ACATGAGAAT 120 TCAGTGTCCC CACTCAGTCA ACTTCGGGGA AACAAGTGTG TCAGGCTATA CAGAACTGCC 180 CCCACTCTCA TTACAGGAGG CTGAACAGCT GGTGCCAGAG AGCTCATGCA ACATGGACAA 240 CCACCAATCA CTCTCAACAA TAAATAAATT AACCAAGGTC ATATGGCGGA AAAAAGCAAA 300 TCAGGAATCA GCAAACCAGA ATTCATTTGA AGTTGTGGAG AGTGAAGTCA GCTTTAAAGG 360 GTTGTGTATG TTAAAGCATA GAATGGTTGA AGAATCATAT AGAAATAGGA GATCAGTAAT 420 CTATTATGAT CTAGCTGGTA ATAGTACATT CTGTAAGCCA ACTGTTTACA TGATCGTTCC 480 TATACATGCA TGCAACATGA TGAAAAGCTG CTTAATTGGC CTTGGTCCCT ACAGAATCCA 540 GGTTGTCTAT GAAAGGACAT ACTGCACTAC AGGTATATTG ACAGAAGGAA AATGCTTTGT 600 TCCTGACAAG GCTGTTGTCA GTGCATTGAA GAGAGGCATG TATGCCATAG CAAGCATAGA 660 GACAATCTGC TTTTTTATTC ATCAGAAAGATAGTAAATAATA GA CTGCCATTAC 720 ATCGGCAATG GGCTCGAAAT GTAATAATAC AGATACTAAA GTTCAAGGAT ATTATATCTG 780 TATTATTGGT GGAAACTCTG CCCCTGTATA TGCCCCTGCT GGTGAAGACT TTAGGGCAAT 840 GGAGGTTTTT TCTGGGATTA TTACATCACC ACATGGAGAA GACCATGACC TCCCTGGCGA 900 AGAAATTGCA ACATACCATA TTTCAGGGCA GATAGAGGCA AAAATCCCTC ATACAGTGAG 960 CTCCAAGAAC TTAAGATTGG CTGCTTTTGC AGGTATTCCA TCATACTCAT CAACTAGTAT 1020 ATTGGCTGCT TCAGAAGATG GTCGTTTCAT ATTTAGTCCT GGTCTATTTC CTAACCTAAA 1080 TCAGTCAGTC TGTGACAACA ATGCACTCCC TTTAATCTGG AGGGGCCTAA TTGATTTAAC 1140 TGGATACTAT GAGGCAGTCC ACCCTTGTAA TGTATTCTGT GTCTTATCAG GACCAGGTGC 1200 TTCATGTGAA GCCTTTTCAG AAGGAGGTAT TTTCAATATT ACTTCTCCAA TGTGCCTGGT 1260 GTCCAAGCAG AATAGGTTTA GAGCAGCTGA GCAGCAGATC AGCTTTGTCT GCCAAAGGGT 1320 TGATATGGAT ATTATAGTGT ACTGTAATGG TCAGAAAAAA ACAATCTTAA CAAAAACATT 1380 AGTTATAGGC CAATGCATTT ATACTATTAC AAGTCTCTTT TCACTGTTAC CAGGGGTTGC 1440 CCATTCTATT GCTATTGAGT TGTGTGTTCC AGGGTTTCAT GGCTGGGCCA CAGCTGCACT 1500 TTTGATCACA TTCTGCTTTG GCTGGGTATT GATTCCTGCA TGTACATTAG CTATTCTT TT 1560 AGTTCTTAAG TTTTTTGCAA ATATCCTTCA TACAAGCAAT CAAGAGAACC GATTCAAAGC 1620 CATTCTACGG AAAATAAAGG AGGAGTTTGA AAAAAGAAAG GGTTCCATGG TTTGTGAGAT 1680 CTGTAAGTAT GAGTGTGAAA CATTAAAAGA ATTGAAGGCA CATAATCTAT CATGTGTTCA 1740 AGGAGAGTGC CCATATTGCT TTACCCACTG TGAACCGACA GAAACTGCAA TTCAGGCACA 1800 TTACAAAGTT TGTCAGGCCA CCCACCGATT CAGAGAAGAT TTAAAAAAGA CTGTAACTCC 1860 TCAAAACATT GGGCCTGGCT GTTACCGCAC ATTAAATCTT TTTAGGTATA AGAGTAGGTG 1920 TTATATTCTG ACAATGTGGA CTCTTCTTCT AATTATTGAA TCCATCCTCT GGGCAGCAAG 1980 TGCAGCAGAA ATCCCCCTTG TCCCGCTCTG GACAGATAAT GCTCATGGCG TTGGGAGTGT 2040 TCCTATGCAT ACAGATCTTG AATTAGACTT TTCTTTGCCA TCCAGTTCTA AGTACACATA 2100 TAAAAGACAT CTCACAAACC CAGTTAATGA CCAACAGAGT GTCTCATTGC ACATAGAAAT 2160 TGAAAGTCAA GGCATTGGTG CTGATGTTCA TCATCTTGGA CATTGGTATG ATGCAAGACT 2220 GAATCTAAAA ACCTCATTTC ATTGTTATGG TGCCTGCACA AAATATCAAT ATCCATGGCA 2280 CACTGCAAAA TGCCATTTTG AGAAAGATTA TGAGTATGAA AATAGCTGGG CATGCAACCC 2340 CCCAGATTGC CCAGGGGTTG GTACAGGTTG TACTGCTTGT GGATTATATC TAGATCAATT 240 0 GAAGCCGGTA GGAACAGCCT TCAAAATTAT AAGTGTAAGA TACAGTAGAA AAGTGTGCGT 2460 GCAGTTTGGT GAAGAACACC TTTGTAAAAC AATTGATATG AATGATTGCT TTGTGACTAG 2520 GCATGCCAAA ATATGTATAA TTGGGACTGT ATCTAAGTTT TCTCAAGGTG ACACTCTACT 2580 GTTCCTAGGG CCTATGGAAG GAGGTGGTAT AATCTTTAAA CACTGGTGCA CATCAACCTG 2640 TCATTTTGGA GACCCTCGTG ATGTCATGGG TCCAAAAGAT AAACCATTTA TTTGCCCTGA 2700 ATTCCCAGGG CAATTCAGGA AAAAATGTAA CTTTGCCACG ACTCCAGTTT GTGAATATGA 2760 TGGAAACATT ATCTCAGGTT ATAAGAAAGT TCTTGCAACA ATTGATTCTT TCCAATCATT 2820 TAATACAAGC AACATACACT TCACTGATGA GAGAATTGAA TGGAGAGACC CTGATGGTAT 2880 GCTCCGGGAT CATATTAATA TTGTTATTTC TAAAGATATT GATTTTGAAA ATTTGGCTGA 2940 GAATCCTTGT AAAGTAGGGC TTCAGGCAGC AAACATAGAA GGTGCCTGGG GTTCAGGTGT 3000 CGGGTTTACA CTCACATGTC AGGTGTCTCT CACAGAATGC CCAACATTTC TCACATCAAT 3060 AAAGGCCTGT GACATGGCAA TTTGTTACGG TGCAGAAAGT GTAACACTCT CACGAGGACA 3120 AAATACTGTC AAAATTACTG GGAAAGGTGG CCATAGTGGT TCTTCATTTA AATGCTGTCA 3180 TGGGAAAGAA TGTTCATCAA CTGGCCTCCA AGCCAGTGCA CCACATCTGG ATAAGGTAAA 3240 TGGT ATCTCT GAGTTAGAAA ACGAGAAAGT TTATGATGAT GGTGCACCTG AATGTGGCGT 3300 TACTTGTTGG TTTAAAAAAT CAGGTGAATG GGTTATGGGT ATAATCAATG GAAACTGGGT 3360 TGTCCTAATT GTCTTGTGTG TCCTGCTGCT CTTTTCTCTT ATCCTGTTGA GCATCTTGTG 3420 TCCTGTTAGA AAGCATAAAA AATCATAAAT CCTACCTATT ATTCTTCACA GCATGTATTG 3480 AGTTTTAAAC ACTTTACCAT TAAAAACTTA ACCTGGCTCT AATATCTGAT AACTAACTTT 3540 CATTTTTATT TTTATATGGA TTAATTACTA AAAAAAATAC TCTCTTCTAT CTCCCAATCT 3600 TTTATTGATT CACCGGGGTG TTGTCTTGAC ATCCTGCGGA GTCTACTACT A 3651

SEQ ID NO: 11 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GGRAACTCYG CMCCHRTATA TG 22

SEQ ID NO: 12 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CCTTCWGAAA AGGCYTCACA TGA 23

SEQ ID NO: 13 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: TCACCRCATG GRGAAGAYCA TGA 23

SEQ ID NO: 14 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence: RGGGTGGAYW GCYTCRTAGT ATC 23

SEQ ID NO: 15 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CATCYAGTTC YARGTAYACA TA 22

SEQ ID NO: 16 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GCCTAGWYAC AAARCAATCR TTC 23

SEQ ID NO: 17 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: TCMTTTCAYT GTTATGGTGC 20

SEQ ID NO: 18 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: AGGTDYTCYT CMCCRAAYTG 20

SEQ ID NO: 19 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CTTTTCTTTG CCATCCAGTT CTA 23

SEQ ID NO: 20 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GGAACAGTAG AGTGTCACCT TGA 23

SEQ ID NO: 21 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CCTCATTTCA TTGTTATGGT GCC 23

SEQ ID NO: 22 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GCCTAGTCAC AAAGCAATCA TTC 23

SEQ ID NO: 23 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GATATGAATG ATTGYTTTGT 20

SEQ ID NO: 24 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CCATCAGGGT CTYTCCA 17

SEQ ID NO: 25 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence: TGTATAATTG GGACWGTATC TAA 23

SEQ ID NO: 26 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GCAAAGTTAC ATTTYTTCCT 20

SEQ ID NO: 27 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: TGTTAYGGTG CAGARAGTGT RAC 23

SEQ ID NO: 28 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GYGCHCCRTC ATCATAWWCT TT 22

SEQ ID NO: 29 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GRRAAAGGTG GCCATAGTGG TTC 23

SEQ ID NO: 30 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: YCYAWCTCAG ARATHCCATT KAC 23

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location // C12Q 1/48 Z 6807-4B (72) Inventor Kiyozo Asada 3 Seta, Otsu, Shiga Prefecture Chome 4-1-1 Central Research Institute, Tama Shuzo Co., Ltd. (72) Inventor Kazutake Takesako 3-4-1 Seta, Otsu City, Shiga Prefecture Chome Research Institute, Mina Shuzo Co., Ltd. (72) Ikunobu Kato Otsu City, Shiga Prefecture 3-4-1 Seta, Central Research Laboratory, Minami Shuzo Co., Ltd. (72) Inventor Yuji Isegawa 7-43 Hiyoshidai Ichibancho, Takatsuki City, Osaka Prefecture

Claims (3)

[Claims] 1. A method for detecting renal symptomatic hemorrhagic fever virus, which comprises reverse-transcription and subsequent two-step polymerase chain reaction (PCR), the nucleic acids represented by SEQ ID NOs: 1 to 5 and their complementary strand sequences, respectively. A method for detecting renal symptomatic hemorrhagic fever virus, comprising the step of amplifying a part of at least one nucleic acid selected from the group consisting of: 2. A method for detecting renal symptomatic hemorrhagic fever virus in a sample, which comprises the following steps. (A) a step of separating the nucleic acid from the sample; (b) the separated nucleic acid is represented by the nucleic acid fragment represented by SEQ ID NO: 6 in the sequence listing or its mutant sequence, and / or the SEQ ID NO: 7 in the sequence listing. Reverse transcribing with a primer selected from the nucleic acid fragment or its mutated sequence to obtain reverse transcribed DNA, (c) the reverse transcribed DNA is SEQ ID NO: 6 in the sequence listing.
A step of carrying out a first-step PCR to amplify DNA by a combination of the nucleic acid fragment represented by or the mutant sequence thereof and the nucleic acid fragment represented by SEQ ID NO: 7 of the sequence listing or the mutant sequence thereof, and (d) amplified DNA A step of carrying out a second-stage PCR with a combination of the nucleic acid fragment represented by SEQ ID NO: 8 in the sequence listing or its mutant sequence and the nucleic acid fragment represented by SEQ ID NO: 9 in the sequence listing or its mutant sequence to amplify DNA ,
And (e) a step of detecting the amplified DNA. 3. A primer for detecting renal symptomatic hemorrhagic fever virus comprising a nucleic acid fragment represented by each of SEQ ID NOs: 6 to 9 in the sequence listing, or a mutant sequence thereof.