WO2006009260A1

WO2006009260A1 - Method of detecting hepatitis e virus

Info

Publication number: WO2006009260A1
Application number: PCT/JP2005/013513
Authority: WO
Inventors: Tsutomu Kageyama
Original assignee: Bml, Inc.
Priority date: 2004-07-23
Filing date: 2005-07-22
Publication date: 2006-01-26
Also published as: JPWO2006009260A1

Abstract

It is intended to provide a method of detecting a virus which comprises detecting, from a detection subject, a nucleic acid of a base sequence consisting of 10 or more consecutive bases selected from a sequence corresponding to the base sequence represented by SEQ ID NO:1 that is the overlap of the ORF2 region and the ORF3 region in a hepatitis E virus gene, and regarding the subject as positive to the hepatitis E virus in the case where the above nucleic acid is detected; and a detection kit for the embodiment of the method. According to this method, a comprehensive or selective system of detecting HEV at a high sensitivity can be established. This detection method is applicable to blood for transfusion, blood preparations, foods, environmental examinations and so on. Moreover, a genotype-specific detection system is usable in clarifying infection route and epidemiological studies.

Description

Specification

Method for detecting hepatitis E virus

Technical field

[0001] The present invention relates to a method for detecting a virus, and more particularly to a method for detecting a hepatitis E virus.

Background art

[0002] Hepatitis E virus (HE V) is a virus that induces hepatitis E and is classified as family Hepeviridae, genus Hepevirus, and has a small (diameter) diameter of about 7.2kb plus-strand RNA genome. 27-30nm) virus. HEV was first cloned by Reyes et al. In 1990 (Reyes, G. R "MA Purdy, JP Kim, KC Luk, LM Young, KE Fry, and DW Bradley. 1990. Isolation of a cDNA from tHEVirus res ponsible for enterically transmitted non— A, non— B hepatitis. Science.vol.247: 1335-9) o After that, the complete nucleotide sequences of many strains were revealed. HEV is classified into four types (genotypes I to IV) (Schlauder, GG, and I. K. Mushahwar. 2001. uenetic heterogeneity of hepatitis E virus. J Med irol, vol.6 5: 282- 92) Many cases of hepatitis E are caused by drinking water contaminated with viruses, and in developing countries, hepatitis E is often caused by drinking water contaminated with HEV.

A. C, and G. b. Fout. 2002. Development of a molecular method to identify hepatitis E virus in water. J Virol Methods, vol. 101: 175-88).

[0003] In developed countries, many cases of travelers infected with developing countries have been recognized as “imported infectious diseases”, but hepatitis E develops even without a history of travel to these regions. “Domestic cases” have also been observed, and it has become clear that HEV strains from which such strengths are collected are unique “indigenous strains” (Erker, J C, SM Desai, GG Schlauder, GJ Dawson, and IK Mushahwar. 1999. A hepatitis E virus variant fr om the United States: molecular characterization and transmission in cynomolgus m acaques. J Gen Virol, vol.80 (Pt 3): 681- 90; Schlauder, GG, GJ Dawson, JC Erker, PY Kwo, MF Knigge, DL Smalley, JE Rosenblatt, SM Desai, and I. 1998. The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States.JG en Virol, vol. 79 (Pt 3): 447— 56; Takahashi, K. K. Mushahwar. , K. Iwata, N. Watanabe, T. Hatahara, Y. Ohta, K. Baba, and S. Mishiro. 2001. Full-genome nucleotide sequence of a hepa titis E virus strain that may be indigenous to Japan. Virology, vol.287: 9—12; Yamam oto, T., H. Suzuki, T. Toyota, M. Takahashi, and H. Okamoto. 2004. Three male pa tients with sporadic acute hepatitis E in Sendai, Japan, who were domestically infect ed with hepatitis E virus of genotype III or IV. J Gastroenterol, vol.39: 292— 8).

[0004] This virus is transmitted by oral infection and develops after an incubation period of 15 days and 60 days. There are many cases of subclinical infection, and some people do not develop even if infected. Typical symptoms are acute hepatitis with gastrointestinal symptoms such as abdominal pain, fever, and vomiting, and strong jaundice with brown urine appears. After jaundice lasts for 12 to 15 days, it usually recovers one month after onset. HEV viremia appears prior to jaundice, and the virus is excreted in the stool. Like hepatitis A, hepatitis E is not chronic, but there are cases where viremia continues for a long time with excretion in the stool. The fatality rate is 0.5-4.0% in the case of onset. For pregnant women, the fatality rate with a high rate of fulminant hepatitis is as high as 17-33%.

[0005] Recently, even in Japan, cases of death from acute hepatitis E, which are thought to be caused by feeding on wild boar liver, have been reported so far. Some cases have been reported indirectly (Meng, XJ, RH Purcell, PG Halbur, JR Lehman, DM Webb, TS Tsareva, JS Haynes, BJ Thacker, and SU Emerson. 1997. A novel virus in swine is closely related to the human hep atitis E virus.Proc Natl Acad Sci USA, vol.94: 9860- 5; Nishizawa, T "M. Takahash i, H. Mizuo, H. Miyajima, Y. Gotanda, and H. Okamoto. 2003. Characterizati on of Japanese swine and human hepatitis E virus isolates of genotype IV with 99% identit y over the entire genome.J Gen Virol, vol.84: 1245-51; Okamoto, H., M. Takahashi, T Nishizawa, K. Fukai, U. Muramatsu, and A. Yoshikawa. 2001. Analysis of the c omple te genome or indigenous swine hepatitis E virus isolated in Japan. Biochem Biophys Res Commun, vol.289: 929-36) ₀ part of the pig lever, which has been commercially available in Hokkaido The gene for hepatitis E virus was detected (Yazaki, Υ ·, H. Mizuo, M. Takahashi, T. Nishizawa, N. Sasaki, Y. Gotanda, and H. Okamoto. 2003. Sporadic acute or fulmin ant hepatitis in Hokkaido, Japan, may be food-borne, as suggested by the presenc e of hepatitis E virus in pig liver as food.J Gen Virol, vol.84: 2351—7), and a group E via raw deer meat Cases of hepatitis B virus food poisoning have also been reported (Tei, S., N. Ki tajima, K. Ta anashi, and S. Mishiro. 2003. Zoonotic transmission of hepatitis E viru s from deer to human beings. Lancet, vol.362: 371-3), it is highly possible that hepatitis E is a zoonotic disease!

[0006] In addition, so far only type III and IV have been found in swine-derived HEV, and infection has also been established in akage monkeys and chimpanzees, and viremia and viral excretion in feces have also been observed (Meng, XJ, PG Halbur, MS Shapiro, S. Govindarajan, JD Bruna, IK Mush ahwar, RH Pur cell, and SU Emerson. 1998. Genetic and experimental evidence f or cross-species infection by swine hepatitis E virus. J Virol, vol. 72: 9714—21). Human HEV is also known to infect pigs (Meng, XJ, PG Halbur, MS Shapiro, S. Govindarajan, JD Bruna, IK Mushahwar, RH Pur cell, and SU E merson. 1998. Genetic and experimental evidence for cross-species infection by swi ne hepatitis E virus.J Virol, vol.72: 9714-21; Halbur, PG, C. Kasorndorkbua, C. Gilbert, D. Guenette, MB Potters, RH Pur cell, SU Emerson , TE Toth, and XJ Meng. 2001. Comparative pathogenesis of infection of pigs with hepatitis E vir uses recovered from a pig and a human. J Clin Microoiol, vol.39: 918—23).

[0007] In a farm in Ontario, Canada, it was reported in 2001 that more than 80% of 6-month-old pigs were positive for HEV antibodies (Yoo, D "P. Willson, Υ · Pei, Μ. A Hayes, A. Deckert, CE Dewey, RM Friendship, Y. Yoon, M. Gottshalk, C. Yason, and A. uiulivi. 2 001. Prevalence of hepatitis E virus antibodies in Canadian swine berds and identific ation of a novel variant Clin Diagn Lab Immunol, vol.8: 12 13-1219) Swine hepatitis E virus.Volume 8:12 13-1219) A high prevalence of hepatitis E virus antibody in pigs has also been reported in the United States, Australia, etc. F. Li, RJ Love, and DA Anderso n. 1999. Serological evidence for swine hepatitis E virus infection in Australian pig h erds. Vet Microbiol, vol. 68: 95—105; Huang, FF, G. Haqshenas, DK Guenette, P. G. Halbur, SK Schommer, FW Pierson, TE Toth, and XJ Meng. 2002. Det ection by reverse transcription — PC R and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United State s. J Clin Microbiol, vol. 40: 1326-32). In Japan, viruses are more likely to be detected in pigs that are 2 to 3 months of age in the early stages of fattening, but it is reported that they cannot be detected from lactation and those over 6 months of age. From this, it is considered that viremia occurs transiently after infection, and the virus disappears from the body with the appearance of antibodies.

[0008] HEV has also been found in sewage in non-indigenous areas, and there is concern about contamination of HEV to the ocean and shellfish (Pina, S., J. Jofre, SU Emerson, RH Purcell, and R. Girones.

1998. Characterization of a strain of infectious hepatitis E virus isolated from sewag e in an area where hepatitis E is not endemic. Appl Environ Microbiol, vol.64: 4485-8).

[0009] Due to the recent accumulation of HEV genomic nucleic acid sequence information, various HEV detection methods based on this have been established in various research groups. However, it cannot be said that a means for efficiently detecting various side forces of HEV has been well established, and there are still challenges for practical use of HEV detection.

Disclosure of the invention

[0010] The present inventor has examined practical means for detecting HEV, and in a specific region of the HEV gene, the mutant is highly conserved even between mutant strains. There are parts where the effects of mutations are very large, and by using the gene region as a HEV detection index, it was found that HEV can be detected efficiently in various aspects. completed.

[0011] That is, the present invention detects a nucleic acid having a base sequence of 10 bases or more selected from a base sequence corresponding to the base sequence shown in SEQ ID NO: 1 with respect to the detection target, and detects the nucleic acid. In this case, the present invention provides a virus detection method (hereinafter also referred to as the present detection method), wherein the detection target is positive for hepatitis E virus (HEV).

[0012] HEV genotypes are currently classified into four types, GI to GIV, and the base shown in SEQ ID NO: 1 The sequence is the nucleotide sequence of the 5253 to 5434 nt cDNA of HEV (GI) strain “M73218: Genbank No. J [hereinafter, this strain may be referred to as a reference strain (prototype).” This is a region that forms part of the HEV “region where ORF2 and ORF3 regions overlap”.

[0013] This detection method detects differences in HEV genotypes by detecting the base sequence of a specific gene region existing in all genotypes of HEV when detecting HEV in a detection target. It is a virus detection method characterized by being capable of both exceeding detection and detecting HEV for each genotype.

In the present specification, the numbers of individual bases in the base sequence of the HEV gene (SEQ ID NOs: 1 to 48) are in accordance with the Genbank No. shown in FIG. For example, guanine (G), the 5253rd base of the above-mentioned prototype M73218 strain, corresponds to guanine (G), the first base of SEQ ID NO: 1.

[0015] This "base sequence of a specific gene region existing in all genotypes of HEV" corresponds to "a base sequence corresponding to SEQ ID NO: 1" according to the present invention. That is, by detecting the presence of the base sequence corresponding to SEQ ID NO: 1 in the detection target and the Z or its contents, qualitative detection, quantitative detection, and further detection of HEV in the detection target are: By grasping the genotype of the causative virus, it is possible to establish a treatment policy for the detection target provider and specify a virus contamination source.

[0016] The above "corresponding nucleotide sequence" means 1) a sequence corresponding to the above-mentioned SEQ ID NO: 1 in each HEV strain, and 2) an antisense sequence of the sequence. Shall mean. In addition, the nucleic acid constituting the base sequence includes, but is not limited to, oligoribonucleotides and oligodeoxyribonucleotides. Peptide nucleic acids, methylphosphonate nucleic acids, S-oligonucleic acids, morpholyl nucleic acids, etc. Of artificially synthesized nucleic acids.

[0017] The nucleic acid to be detected must be an oligonucleotide selected from the base sequence corresponding to SEQ ID NO: 1 and continuous for at least 10 bases, and the maximum is about 180 bases. In general, it is preferable that the oligonucleotide is continuous for about 15 to 30 bases.

[0018] In addition, the base difference with respect to the base sequence represented by SEQ ID NO: 1 is 10% Must be within. The difference in bases between all genotypes of the conserved region in the HEV “region where ORF2 region and ORF3 region overlap” is within 10%, and the quasi-conserved region (conserved between different genotypes, However, the difference in bases between HEVs of the same genotype in a gene region that is well conserved in the same genotype is also within 10%! /.

[0019] As a detection target of this detection method, an organism individual, for example, any mammal including humans, monkeys, pigs, ushi, and sheep, or oysters, clams, clams, shijimi, mussels, etc. Biological samples such as body fluids, blood, serum, lymph, feces, and tissues collected from marine products, as well as environmental water including seawater, river water, lake water, sewage, drainage, tap water, well water, etc. Can be mentioned. In addition, in food and food production facilities, food itself, deposits such as cooking utensils in food production facilities, deposits such as clothing of food producers, papers and cloths that wipe them, and the like can also be detected. Each detection target can be subjected to appropriate pretreatments such as homogenization, concentration, and extraction as necessary, and the sample obtained in this way can also be used as a test target. is there. As for the method of obtaining genes from these detection targets, it is possible to select an appropriate method according to the type of detection target used. Generally, the detection target to be used is generally immersed or suspended in water or the like. From the supernatant fraction obtained from these waters, viral RNA can be extracted by a conventional method such as the acid phenol method (AGPC method: Acid Guanidin Phenol Chroloform method). For example, by preparing cDNA complementary to viral RNA using reverse transcriptase and using this nucleic acid sample directly or performing gene amplification using gene amplification primers. (In general, confirmation of the desired gene amplification product is carried out based on whether or not a gene amplification product of the desired size is found by electrophoresis.) .

[0020] Examples of gene amplification methods include PCR method, RT-PCR method, real-time PCR method, SDA ^ strand displacement amplification (NASDA) method, NASBA (Nucleic Acid Sequence Based Amplification) method, LAMP (Loop—mediated isothermal amplification) Law. Nucleic acid amplification products can be detected by electrophoresis, electrochemical sensors such as nucleic acid detection chips, gene sensors using quartz crystal, or fluorescently labeled primers or probes. Alternatively, nucleic acid amplification products can be detected as HEV-derived genomic fragments by measuring fluorescence intensity with a fluorescence sensor using a fluorescence intercalation agent. In addition, fluorescent labeling, chemical modification, protein modification, radioisotope labeling, etc. are applied to the probe, and dot hybridization, slot hybridization, Southern hybridization, Northern hybridization method In combination with various amplification methods, etc., TaVMan Probe, Molecular Beacon Probe ^ Nob hybridization probe, analysis by real-time quantification method using LUX (Light Upon extension) primer, etc. Can be detected. Furthermore, HEV genomic fragments can be detected using the above-mentioned oligonucleotides even in methods that do not require amplification of the target gene, such as the invader method.

[0021] In addition, the detection of the target nucleic acid in this detection method is mainly performed by positively or negatively detecting nucleic acid nucleic acids and hybrids, and a specific method thereof is a known detection method. Techniques can be used. For example, dot hybridization, slot hybridization, Southern hybridization, Northern hybridization method, etc., or in combination with various amplification methods, TaqMan probe, molecular beacon probe ( The target HEV-derived genomic fragment can be detected by analysis using a real-time quantification method using molecular beacon probes), noisy hybridization probes, LUX (light upon extension) primers, etc. . Furthermore, the target HEV genome fragment can also be detected by methods that do not require amplification of the target gene, such as the invader method.

[0022] Depending on the specific needs in these techniques, the nucleic acid according to the present invention (used as a probe, a primer, etc.) can be added with necessary modifications, and these modified nucleic acids are also As long as the base sequence is within the scope of the present invention, it is similarly assumed to be within the scope of the present invention. Examples of the additional element include enzyme modification (peroxidase, etc.), fluorescent labeling (luciferin, etc.), chemical modification, protein modification (piotine, etc.), radioisotope labeling, and the like.

[0023] In particular, in order to avoid dangers such as contamination and to reduce the time spent for the detection operation as much as possible, in the course of the gene amplification operation, a specific base sequence in the gene amplification product is used. It is preferred to use a means capable of monitoring the presence of the row. Representative examples of such methods include, for example, the detection method using the above-described molecular beacon probe, the detection method using the Taq-Man Probe, and the LUX (Light Upon Extension). Examples include detection methods using primers.

[0024] The detection method using a molecular beacon probe is a hairpin type that can be used to monitor the formation of gene amplification products by the PCR method or the like during or after the amplification process. This is a method for detecting a gene using a hybridization probe (molecular beacon probe) (Nature Biotechnology 1998 16: 49-53). The ends of the nucleic acid constituting the molecular beacon probe are complementary to each other. Usually, these ends are joined together to form a so-called stem structure, and the loop portion in the striking stem structure is a gene amplification. It is designed to be complementary to the target region of the product. In addition, when the phosphor and the non-fluorescent quencher are bound to both ends of the nucleic acid and released in solution, a hairpin structure is formed, so that the fluorophore and the quencher interact with each other. And the fluorescence disappears. However, when a gene amplification product having a base sequence complementary to the nucleic acid exists, the loop portion binds to the complementary base sequence, and as a result, the structure of the entire probe changes. Since the phosphor and the quencher are separated from each other and the quenching effect of the quencher on the phosphor is eliminated, the phosphor emits the original fluorescence. The increase in fluorescence intensity due to the elimination of this quenching effect is proportional to the increase in gene amplification products having a base sequence complementary to the nucleic acid. By monitoring this increase in fluorescence intensity, the target nucleotide sequence can be detected not only after the gene amplification process but also during the gene amplification process. That is, the target nucleic acid in the detection sample can be detected using the increase in fluorescence intensity as an index.

[0025] It should be noted that in the molecular beacon probe, the above-mentioned fluorescent label and quencher label are usually labeled with 6-carboxyfluorescein (6-FAM) or 6-carboxy-4,7,2 'at the 5th end of the nucleic acid. , 7'-tetrachlorofluorescein (TET) and other fluorescein-based fluorescent dyes and rhodamine-based fluorescent dyes such as 5-carboxytetrame thylrhodamine (TAMARA) are labeled, and at the 3 'end , 4- (4′-dimethylaminophenylazo) benzoic acid (DABC YL) can be used for labeling (for example, Nature Biotechnology 1996 14: 303-308).

[0026] The detection method using the Taq-Man Probe is a hybridization probe that can be used to monitor the formation of a gene amplification product by PCR or the like during the amplification process with fluorescence ( This is a method for detecting genes using Taq-Man Probe (Experimental Medicine Vol.15 No.7 (Special Issue) p46-51, 1997, etc.). The Tac Couman probe is a nucleic acid labeled with 5, a fluorescein fluorescent dye (reporter dye) at the end, and a rhodamine fluorescent dye (taentia dye) at the 3 'end. In a state where the reporter dye and the quencher dye are bound via a nucleic acid, the fluorescence of the reporter dye is suppressed by the quencher dye due to the Forster resonance energy. On the other hand, when the primer and the Tatooman probe anneal the nucleic acid complementary to the Taucman probe nucleic acid of the gene amplification product and the extension reaction proceeds, TaqDNA polymerase 5, → 3, due to the endonuclease activity, Hydrolysis occurs from the 5 'end of the probe, and when the 5' end reporter dye is released from the 3 'end quencher, the fluorescence intensity of the reporter dye increases. The increase in fluorescence intensity by the reporter dye is proportional to the increase in gene amplification products having a base sequence complementary to the nucleic acid. By monitoring this increase in fluorescence intensity, the target base sequence can be detected not only after the gene amplification process but also during the gene amplification process. That is, the target nucleic acid in the detection sample can be detected using the increase in fluorescence intensity as an index.

[0027] It should be noted that the above-described fluorescent labeling in the Tac Couman probe usually has a fluorescein-based fluorescent dye such as 6-FAM or TET at the 5 'end of the nucleic acid and a rhodamine such as TAM ARA at the 3' end. The dye of the system can be carried out according to a conventional method (for example, Nucleic Acids Research 1993 21 (16): 3761-3766).

[0028] The detection method using a LUX (Light Upon Extension) primer is a method of detecting the amplification of a target gene by monitoring the fluorescence during the amplification process for the formation of gene by-products by PCR or the like. is there. The LUX primer is a nucleic acid designed to label a single fluorophore near the 3 'end and take a hairpin structure between the 5' end. Length is through Usually it is 20 to 30 bases, and when the primer has a hairpin structure, it has a quenching ability and does not emit fluorescence, but when it is incorporated into a primer-stranded PCR product, the quenching is released and the fluorescence signal increases. By measuring this increase in signal, the amplified gene product of interest is quantitatively analyzed (Nazarenko, I. et al. (2002) Nucleic Acids Research 30: e37.).

[0029] All the above-described detection nucleic acid probes or primers can be used as detection nucleic acid probes that can be used as components of the detection kit described later.

[0030] HEV detection in the present detection method can be performed by quantifying and detecting a target nucleic acid by the above-described means, such as detection of a gene amplification product having a target base sequence. For example, it can be detected as qualitative information of positive or negative without quantification. HEV can be detected by using detection information (quantitative values and qualitative information) such as gene amplification products as an index and correlating it with the presence / absence of HEV in the sample, and further with the abundance.

Brief Description of Drawings

[0031] FIG. 1 is a drawing showing the results of examining the diversity of HEV genomes.

FIG. 2 is a drawing showing the strain lines in the HEV genome.

FIG. 3 shows a parallel comparison of the base sequences in each strain of the HEV gene region used as a detection region in the present invention. In FIG. 3, the three base sequences in the second row are the base sequences of SEQ ID NOs: 49, 51 and 57 from the left. In Fig. 3, the base sequence from the 3rd row to the 30th row is also the upper force, SEQ ID NO: 1, 15, 13, 10, 12, 17, 16, 11, 7, 8, 6, 4, 3, 2, 5, 9, 14, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 and 58.The base sequences of the 32nd and 33rd lines are respectively , SEQ ID NOS: 18 and 53, and the base sequences of the 35th to 45th rows are SEQ ID NOS: 21, 27, 26, 25, 24, 28, 23, 22, 19 from the top, respectively. , 56 and 54, and the base lines in the 47th to 57th lines, Ui, Ue, et al., Respectively, Umi 37, 31, 32, 36, 33, 34, 35 , 30, 29, 38 and 55.

BEST MODE FOR CARRYING OUT THE INVENTION

[0032] Here, in order to clarify the significance of the nucleotide sequence shown in SEQ ID NO: 1, gene analysis of HEV will be described. [0033] Determination of HEV strain gene type

37 existing HEV strains with almost full-length nucleotide sequences have been identified (see below for accession numbers). The type was determined according to the existing classification.

[0034] <Genetic type of HEV strain with almost existing full-length sequence>

Genotype I (GI)

M80581 (SEQ ID NO: 2) .L25595 (SEQ ID NO: 3), L25547 (SEQ ID NO: 4), M94177 (SEQ ID NO: 5), L08816 (SEQ ID NO: 6), D11092 (SEQ ID NO: 7), D11093 (SEQ ID NO: 8), X98292 (SEQ ID NO: 9), AF185822 (SEQ ID NO: 10), M73218 (SEQ ID NO: 1), D10330 (SEQ ID NO: 1 1), AF459438 (SEQ ID NO: 12), AF076239 (SEQ ID NO: 13), X99441 (SEQ ID NO: 14), A F051830 (SEQ ID NO: 15), AY230202 (SEQ ID NO: 16), AY204877 (SEQ ID NO: 17)

Early transmission 11 (GTT)

M74506 (SEQ ID NO: 18)

Early transmission TIT (GTTT)

AF455784 (SEQ ID NO: 19), AY115488 (SEQ ID NO: 20) *, AF082843 (SEQ ID NO: 21) *, AF060669 (SEQ ID NO: 22), AF060668 (SEQ ID NO: 23), AB089824 (SEQ ID NO: 24), AB 074920 (SEQ ID NO: 25) ), AB074918 (SEQ ID NO: 26), AB073912 (SEQ ID NO: 27) *, AB09 1394 (SEQ ID NO: 28)

Early transmission TV (GTV)

AJ272108 (SEQ ID NO: 29), AB108537 (SEQ ID NO: 30), AB074917 (SEQ ID NO: 31), AB0 80575 (SEQ ID NO: 32), AB097811 (SEQ ID NO: 33) *, AB097812 (SEQ ID NO: 34), AB099 347 (SEQ ID NO: 35) ), AB091395 (SEQ ID NO: 36), AB074915 (SEQ ID NO: 37)

* Indicates HEV strains derived from pigs, others are derived from humans.

[0035] <Production of oligonucleotides>

Next, within the shortest possible HEV gene base sequence (specifically, within about 250 nt), 1) 5'-side HEV common primer, 2) HEV common probe, or 3) each genotype HE In order to find a region where V-specific probes, 4) each genotype HEV-specific probe, or 5) HEV common probe, 6) 3 'HEV common primer can be designed, the full-length genome is determined. Sliding Window Analysis (Proutski, V., and E. Holmes. 1998. SWAN: sliding window analysis of nucleotide sequence variability.Bio informatics, vol.l4: 467-8) (Window size: 30, shift: 6) (Fig. 2). As a result, it was found that the location that best met the conditions in the HEV genome was in the area shown in gray in Fig. 1 (corresponding to 5250-5440 nt in M73218), and the range was approximately 180 nt. Using the base sequences of HEV strains (37 strains with almost full-length base sequence revealed and 11 strains with partial sequences known below) whose regions corresponding to this are known, Figure 3 shows the alignment of each type. In FIG. 3, the white box region is a region having a sequence common to all strains, and oligonucleotides serving as a common primer and a common probe were designed here. In addition, the gray box region is a region having a sequence common to only each genotype, and an oligonucleotide that is a probe specific to each genotype was designed here. As a condition for selecting each gene-specific probe, “a nucleic acid having a base sequence corresponding to SEQ ID NO: 1 has at least 11 base groups out of 14 consecutive bases stored for each genotype, and The region is a region that differs by at least 5 bases from the corresponding base sequence of other genotypes. The designed oligonucleotide sequence is shown below.

[0036] <Understanding the partial nucleotide sequence!

AB075971 (SEQ ID NO: 38), AF051349 (SEQ ID NO: 39), AF051350 (SEQ ID NO: 40), AF 051351 (SEQ ID NO: 41), AF051352 (SEQ ID NO: 42), AF058684 (SEQ ID NO: 43), AF065 061 (SEQ ID NO: 44) , AF124406 (SEQ ID NO: 45), AF124407 (SEQ ID NO: 46), M32400 (SEQ ID NO: 47), U22532 (SEQ ID NO: 48)

[0037] <Designed oligonucleotide>

HEV co-sense primer:

(Equivalent to GLM73218 5257-5276 nt)

HECOM-S primer (5, -CGGCGGTGGTTTCTGGRGTG-3 ,: SEQ ID NO: 49) HEV co-sense primer:

(Equivalent to GLM73218 5427-5399 nt)

HECOM-AS primer (5——GGGCGCTKGGMYTGRTCNCGCCAAGNGGA—3: SEQ ID NO: 50) HEV common probe:

(Equivalent to GLM73218 5305-5334 nt)

TP-HECOM probe (5,-(FAM) -CCCCYATATTCATCCAACCAACCCCTTYG C- (TAMRA) -3 ': SEQ ID NO: 51)

HEV gene type I specific probe:

(Equivalent to GLM73218 5332-5350 nt)

TP-HEG1 probe (5, — (FAM) — CGCCCCSRATGTCACCGCT— (TAMRA) — 3 ,: Sequence number 52)

HEV gene type II specific probe:

(Corresponds to GLM73218 5329-5352 nt and corresponds to GILM74506 5229-5332 nt) TP-HEG2 probe (5,-(FAM) -CTTTGCCCCAGACGTTGCCGCTGC- (TAMRA) -3 ': SEQ ID NO: 53)

HRV transmission 111 probe:

(Corresponds to GLM73218 5343-5365 nt and corresponds to GIILAF082843 5367-5389 nt) TP-HEG3 probe (5, — (FAM) -TCGTTTCACAAYCCGGGGCTGGA- (TAMRA) -3 ': SEQ ID NO: 54)

HRV transmission IV probe:

(Corresponds to GLM73218 5330-5352 nt and corresponds to GIV—AB097811 5371-5393 nt) TP-HEG4 probe (5, — (FAM) -TTCGCATCTGACATWCCARCCGC- (TAMRA) -3 ′: SEQ ID NO: 55)

[0038] <Generation of each genotype standard>

Next, a standard control of each genotype having this region was prepared. GI: M 73218 corresponds to 5253-5432 nt, GII: M74506 corresponds to 5223-5402 nt, GIII: AF08284 3 corresponds to 5277-5456 nt, GVI: AB097811 corresponds to 5294-5473 nt (180nt) A plasmid (pT7Blue vector) having γ was prepared and used as a standard for each genotype of HEV.

[Usage mode in this detection method]

As described above, the oligonucleotides of the nucleotide sequences shown in SEQ ID NOs: 49 to 55 (complementary strands) The oligonucleotide having a base sequence of 10 bases or more that is selected from base sequences having 90% or more homology with each base sequence is an oligonucleotide within the scope of the present invention. Based on this premise, the usage of SEQ ID NOS: 49 to 55 in this detection method will be described.

[0040] (1) When detecting HEV beyond genotype

In this case, in the above embodiment, it is necessary to detect the nucleic acid in the conserved region conserved in each genotype of HEV. In the above example, the HEV common probe (SEQ ID NO: 51) Nucleic acid corresponding to a nucleic acid having a base sequence of 10 bases or more selected from base sequences having a homology of at least%.

[0041] First, when a target nucleic acid is detected by a hybridization reaction without performing nucleic acid amplification, the oligonucleotide of SEQ ID NO: 51 is used as a probe for the nucleic acid sample derived from the detection target.ヽ If the hybridizing reaction is positive, it is possible to make the detection target HEV positive (genotype is not yet determined). The oligonucleotide probe having the base sequence of SEQ ID NO: 51 can be labeled appropriately depending on the detection mode.

[0042] Further, an amplification product of a nucleic acid in a nucleic acid sample derived from the detection target is detected using the molecular weight of the gene fragment directly appearing on electrophoresis as an index without performing a nucleic acid hybridization reaction, When an amplification product of the target gene region is detected, it is possible to make the detection target HEV positive (genotype is not determined). In this case, as a primer used for the amplification reaction of nucleic acid, it is possible to use the primer of SEQ ID NO: 49 or 50. The sequence described as the probe of SEQ ID NO: 51 is used as a primer. Is also possible. That is, the primer oligonucleotide sets that can be used in this embodiment are the oligonucleotide set of SEQ ID NOs: 49 and 50, the set of base sequences of SEQ ID NOs: 49 and 51, and the set of base sequences of SEQ ID NOs: 50 and 51. A group is applicable. The oligonucleotide primers having the nucleotide sequences of SEQ ID NOs: 49 to 51 can be appropriately labeled depending on the detection mode.

[0043] Further, when a nucleic acid hybridization reaction is performed on a nucleic acid amplification product in a nucleic acid sample derived from a detection target, and HEV is detected using the hybridization reaction as an index The oligonucleotide of SEQ ID NO: 51 as a probe is subjected to a hybridization reaction with a nucleic acid sample subjected to an amplification reaction derived from the detection target. If the hybridization reaction is positive, HEV is detected in the detection target. It can be positive (genotype is undetermined). In this case, as a primer used for the nucleic acid amplification reaction, it is possible to use the primer of SEQ ID NO: 49 or 50 as well as the oligonucleotide of SEQ ID NO: 51 as a primer. . That is, the probe that can be used in this embodiment is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 51, and the set of primer nucleic acids is the set of oligonucleotides having the nucleotide sequences of SEQ ID NO: 49 and 50, SEQ ID NO: 49 and 51 This corresponds to the base sequence set of SEQ ID NOs: 50 and 51. The probe of SEQ ID NO: 51 and the oligonucleotide primer of the base sequence of SEQ ID NOs: 49 to 51 can be appropriately labeled depending on the detection mode.

[0044] (2) When HEV is detected for each genotype

On the other hand, the need to detect HEV for each genotype is also extremely important, for example, when identifying contamination sources. In this case, instead of the HEV common probe described above, an individual using a nucleotide sequence that is specific for each of the GI to GIV gene types and has a highly conserved base sequence within the same genotype. Use a probe. In the above example, H EV (GI) is the probe of SEQ ID NO: 52, HEV (GII) is the probe of SEQ ID NO: 53, HEV (GIII) is the probe of SEQ ID NO: 54, and HEV (GIV ) Is the probe of SEQ ID NO: 55.

[0045] First, when a target nucleic acid is detected by a hybridization reaction without nucleic acid amplification, the oligonucleotides of SEQ ID NOs: 52 to 55 are selected according to the genotype (GI to GIV) of the HEV to be detected. Using a nucleotide individually as a probe, a hybridization reaction is performed on a nucleic acid sample derived from the detection target, and if the hybridization reaction force is positive for any of the genotype probes, the detection target is not detected. Thus, HEV can be positive in the genotype. The oligonucleotide probes having the nucleotide sequences of SEQ ID NOs: 52 to 55 can be appropriately labeled depending on the detection mode.

[0046] In addition, an amplified product of a nucleic acid in a nucleic acid sample derived from a detection target is converted into a nucleic acid hybrid. If the amplified product of the target gene region is detected using the molecular weight of the gene fragment that appears directly on electrophoresis as an index It is possible to make the target genotype HEV positive. In this case, as a primer used for the nucleic acid amplification reaction, it is preferable to use the oligonucleotides described as probes of SEQ ID NO: 49 or 50 and SEQ ID NOs: 52 to 55 as primers. That is, the primer oligonucleotide pair that can be used in this embodiment is the oligonucleotide pair (GI) having the nucleotide sequence of SEQ ID NO: 49 or 50 and SEQ ID NO: 52, or the oligonucleotide having the nucleotide sequence of SEQ ID NO: 49 or 50 and SEQ ID NO: 53 Group (GII), SEQ ID NO: 49 or 50 and oligonucleotide group (GIII) with the nucleotide sequence of SEQ ID NO: 54, and SEQ ID NO: 49 or 50 and oligonucleotide group (GVI) with the nucleotide sequence of SEQ ID NO: 55 . The oligonucleotide primers having the nucleotide sequences of SEQ ID NOs: 49 to 50 and 52 to 55 can be appropriately labeled depending on the detection mode.

In addition, when nucleic acid hybridization is performed on a nucleic acid amplification product in a nucleic acid sample derived from the detection target, and HEV is detected by genotype using the hybridization reaction as an indicator, the detection purpose is used. When a hybridization reaction is performed on a nucleic acid sample subjected to an amplification reaction derived from a detection target using an oligonucleotide of SEQ ID NO: 52 to 55 as a probe according to the genotype of HEV, and the hybridization reaction is positive In addition, it is possible to make the detection target genotype HEV positive in the detection target. In this case, as a primer used for the nucleic acid amplification reaction, it is possible to use the primer of SEQ ID NO: 49 or 50 as well as the oligonucleotide of SEQ ID NO: 52 to 55 as a primer. is there. That is, the probe that can be used in this embodiment is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 52 when the detection genotype is GI, and the primer nucleic acid pair is an oligonucleotide having the nucleotide sequences of SEQ ID NOs: 49 and 50 And a pair of oligonucleotides having the nucleotide sequences of SEQ ID NOs: 49 and 52, and a pair of oligonucleotides having the nucleotide sequences of SEQ ID NOs: 50 and 52. When the detection genotype is GII, the oligonucleotide having the nucleotide sequence of SEQ ID NO: 53 is the probe, and the primer nucleic acid pair is the oligonucleotide pair of SEQ ID NOS: 49 and 50, the sequence number 49 A set of oligonucleotides with base sequences of 53 and 53, base sequences of SEQ ID NOs: 50 and 53 The set of oligonucleotides is applicable. When the detection genotype is GIII, the oligonucleotide having the nucleotide sequence of SEQ ID NO: 54 is the probe, and the primer nucleic acid pair is the oligonucleotide pair of SEQ ID NOS: 49 and 50, SEQ ID NO: 49 And oligonucleotide pairs having the nucleotide sequences of 54 and 54, and oligonucleotide pairs having the nucleotide sequences of SEQ ID NOS: 50 and 54 are applicable. When the detection genotype is GIV, the oligonucleotide having the nucleotide sequence of SEQ ID NO: 55 is the probe, and the primer nucleic acid pair is the oligonucleotide pair of SEQ ID NOS: 49 and 50, SEQ ID NO: 49 This includes a pair of oligonucleotides having a base sequence of 55, and a pair of oligonucleotides having the base sequences of SEQ ID NOS: 50 and 55.

[0048] [This detection kit]

The present invention is also an invention that provides a detection kit (this detection kit) for carrying out this detection method.

[0049] In this detection kit, 1) an amplification primer for amplifying the whole or a part of the nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO: 1 of HEV [in the above example, typically, Oligonucleotides having the nucleotide sequences of SEQ ID NOs: 49 to 50, provided that the oligonucleotides shown as common probes of SEQ ID NO: 51 (when performing detection exceeding genotypes) or specific probes of SEQ ID NOs: 52 to 55 As described above, the oligonucleotide (when detecting for each genotype) can also be used as a primer.] Or 2) Hybridize to the nucleic acid of a specific HEV to be detected. A probe for detecting the nucleic acid (a common probe of SEQ ID NO: 51 for detection exceeding genotype, or a specific sequence of SEQ ID NO: 52 to 55 for detection of each genotype) Professional B) is included depending on the specific detection method.

In addition to this, for example, specific objects used for detection, for example, a nucleic acid detection instrument (a DNA chip, a substrate provided with a plurality of wells), a reagent used in a detection method used, a label A kit may be formed by combining necessary reagents such as a reaction vessel and a reaction buffer in combination with substances, enzymes, and the like. However, the present detection kit is not limited to the above combinations, and can be provided in combination with various types as necessary.

[0051] This detection kit, which is considered to be the most typical, has the following minimum configuration: It is.

[0052] 1) A nucleic acid having a nucleotide sequence having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 49, and a nucleic acid having a nucleotide sequence having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 50. HEV gene amplification primer set.

[0053] 2) Probe nucleic acid having a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 51, and Z or having 90% or more homology with the base sequence shown in SEQ ID NOs: 52 to 55 A probe nucleic acid set having a base sequence.

Example

[0054] Examples of the present invention will be described below, but the scope of the present invention should not be limited by these examples.

[0055] [Detection method using HEV common probe]

Using a standard for each genotype, a detection method using a common probe was examined. First, using the HEV common sense primer of SEQ ID NO: 49, the set of HEV common antisense primer of SEQ ID NO: 50, and the HEV common probe of SEQ ID NO: 51, the detection sensitivity for each gene type standard was determined. Specifically, using TaqMan Universal Buf fer Kit (ABI, USA), reaction solution (Buffer 25 μ1, HECOM-S primer 500nM, HE COM-AS primer 500nM, TP-HECOM probe 5-20pmol, each standard DNA Prepare 5-5 x 10 ⁷ copies, adjusted with sterile distilled water to a total volume of 50 μl), and perform PCR reaction at ΑΒΙ7900 (ΑΒΙ, USA) (PCR cycle is 50 ° C for 2 minutes → 95 ° C) The fluorescence intensity of 10 minutes → (95 ° C. for 15 seconds → 56 ° C. for 1 minute) × 50 cycles) was measured over time. Standard DNA was prepared separately for each genotype. As a result, all genotype standards DNA can be detected from 5 copies / reaction, and by referring to the Ct value (threshold cycle number), HEV standards of all genotypes are quantitative regardless of genotype. It was apparent that it was detectable.

[0056] [Examination of detection method using each HEV genotype specific probe]

The detection method using each genotype-specific probe was examined using each genotype standard. First, the HEV common sense primer of SEQ ID NO: 49, the set of HEV common antisense primer of SEQ ID NO: 50, and each HEV of SEQ ID NO: 52 to 55 Detection sensitivity for each genotype standard was determined using a gene-type specific probe individually. Specifically, using TaqMan Universal Buffer Kit (ABI, USA), reaction solution (Buf fer 25 μ 1, HECOM—S primer 500nM ゝ HECOM— AS primer 500nM ゝ TP— HEG KTP-HEG2, TP-HEG3 or TP -HEG4) Probe 5 to 20 pmol, each standard DNA 5 to 5 X 10 ⁷ copies, prepared with sterilized distilled water to a total volume of 50 μ1), and prepare ABI7900 (ABI, USA) during PCR reaction (PCR As for the cycle, the fluorescence intensity of 50 ° C. for 2 minutes → 95 ° C. for 10 minutes → (95 ° C. for 15 seconds → 56 ° C. for 1 minute) × 50 cycles) was measured over time. Standard DNA was prepared separately for each genotype. As a result, when TP-HEG1 was used as the genotype-specific probe, only the genotype GI standard was used. When TP-HEG2 was used, only the genotype GII standard was used, and TP-HEG3 was used. Recognizes only the genotype GIII standard, and when TP-HEG4 is used, it specifically recognizes only the genotype GIV standard (each does not cross-react with other genotype standards). It was clarified that detection was possible using standard 5 copies / reaction specifically, and that the HEV standard of each genotype could be detected quantitatively by referring to the Ct value (threshol d cycle number).

That is, when the HEV common probe of SEQ ID NO: 51 is used as the probe, all HEVs can be detected with high sensitivity and quantitative regardless of the gene type. In addition, when using the specific probe TP-HEG1 of SEQ ID NO: 52 as the probe, only GI HEV is used as the probe, and only Gil HEV is used as the probe when using the specific probe TP-HEG2 of SEQ ID NO: 53, When the specific probe TP-HEG3 with SEQ ID NO: 54 is used as the probe, only GIII HE V is quantitatively detected, and when the specific probe TP-HEG4 with SEQ ID NO: 55 is used as the probe, only GI V HEV is quantitatively detected. This can be demonstrated by the above examples. In this case, there is no reaction at all except the genotype recognized by the probe used, so that highly sensitive quantitative detection can be performed specifically for each genotype. The primer sets (HECOM-S, HECOM-AS) used in all reactions are the same.

[0058] [Studies using stool and serum samples obtained from a model of HEV infection in the Japanese macaque]

In 1998, infectious HEV was prepared from the stool of a patient suffering from hepatitis E in India and injected into a Japanese macaque. Fecal stool 10 days after infection, serum before HEV infection and Serum serum from 10 days to 60 days after HEV infection (total of 6 points every 5 days for the first 10-30 days after the first infection and 4 points every 8 days for the remaining 31-60 days) The HEV was detected using this method. First, the stool specimen was prepared with 10 mM Phos phate buffered saline (PBS) so as to be 10% (w / v). RNA extraction from stool samples and serum samples was performed using the QIA Viral RNA Kit (QIAGEN, USA). Next, reverse transcription reaction was performed for each RNA sample. Reverse transcription reaction was performed with each RNA sample (81), reverse transcription reaction solution 12 1 (1 μl of 10 mM dNTP solution, 75 pmol random hexamer, 3 Ounits RNAsin (Promega, USA), 200 units Superscript II RNAseH ( ―) Reverse- transcriptase (Invitrogen, USA), lOOmM DTT 1 μ 1 and 5x reverse transcription buffer (250 mM Tris-Hcl (pH 8.3), 375 mM KC1, 15 mM MgC12) After dilution with sterile distilled water and reaction at 42 ° C for 1 hour or longer, the enzyme was inactivated at 99 ° C for 5 minutes to prepare cDNA (RT Products) for each RNA sample. For PCR reaction, TaqMan Universal Buffer Kit (ABI, USA) is used, and the reaction solution (Buffer 25 μ1, HECOM-S primer 500nM ゝ HECOM-AS primer 500ηM, TP-HECOM probe 5-20pmol, total volume 45μ1) (Adjusted with sterilized distilled water) and 5 μl of RT Products were mixed, and the fluorescence intensity during the PCR reaction was measured over time under the conditions described above. As a result, HEV was detected at 6 points from feces and 10-30 days after infection, and it was strong not detected from serum before HEV infection and serum after 31 days after infection. This result is the result of nested PCR in other regions (Li, T. C, Y. Suzaki, Y. Ami, TN Dhole, T. Miyamura, and N. Takeda. 2004. Protection of cynomolgus monkeys against HEV inf ection by oral administration of recombinant hepatitis E virus-like particles.Vaccine, vol.22: 370-7), very sensitive and specific HEV detection from serum or feces from HEV viremia It became clear that this is possible. When each genotype-specific probe was used, the reaction mixture (Buffer 25 1, HECOM-S primer 500 nM ゝ HECOM-AS primer 500 nM ゝ (TP- HEG1, TP- HEG2, TP- HEG3 or TP- HEG4 ) Probe 5 to 20 pmol, RT Products 2 μ1 adjusted with sterile distilled water to a total volume of 50 μ1, and measured the fluorescence intensity during the PCR reaction over time under the conditions described above. All did not react forcefully with the TP-HEG1 probe. That is, all of these HEV genotypes were determined to be GI. [0059] [Examination using plasmids with full-length GI and GIV HEV genomic sequences] Whether or not HEV can be detected correctly with this detection system, even if the sequence is longer, using sequences that are longer than each genotype standard. We also examined whether genotype-specific detection was possible. As a result of investigating this detection method using plasmids with full-length GI and GIV HEV genomic sequences, both copies were able to detect 5 copies / reaction force quantitatively using the common probe TP-HECOM. For genotype-specific detection, the GI plasmid uses only TP-HEG1, a HEV genotype I-specific probe, and the GEV plasmid uses TP-HEG4, a HEV genotype IV-specific probe. There was no reaction, and in this case too, each genotype was specific and could be detected quantitatively by 5 copies / reaction.

Industrial applicability

[0060] According to the present invention, a comprehensive or selective HEV high-sensitivity detection system can be established.

The HEV detection method of the present invention can be used for blood for blood transfusion, blood products, foods, environmental tests, etc., and the genotype-specific detection system can be used for elucidation of infection routes, epidemiological studies, and the like.

Claims

The scope of the claims

[1] When a nucleic acid having a base sequence of 10 bases or more selected from the base sequence corresponding to the base sequence shown in SEQ ID NO: 1 is detected and the nucleic acid is detected, A method for detecting a virus, wherein the detection object is positive for hepatitis E virus.

[2] Nucleic acid having a base sequence of 10 bases or more selected from the base sequence corresponding to the base sequence shown in SEQ ID NO: 1 is detected by detecting a hybrid between the nucleic acid and the nucleic acid in the detection target. The method for detecting a virus according to claim 1.

[3] The nucleic acid in the detection target is used as a nucleic acid obtained by amplifying a nucleic acid having a base sequence of 10 or more consecutive bases selected from the base sequence corresponding to the base sequence shown in SEQ ID NO: 1. The detection method of the virus as described.

[4] Nucleic acid having a base sequence of 10 bases or more selected from the base sequence corresponding to the base sequence shown in SEQ ID NO: 1 is detected by detecting a hybrid between the nucleic acid and the nucleic acid in the detection target. The virus detection method according to claim 1, wherein the nucleic acid is a nucleic acid obtained by performing an amplification treatment.

[5] The nucleic acid obtained by performing the amplification treatment is a nucleic acid having a base sequence of 10 bases or more selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 49, and SEQ ID NO: 50 Selected from a nucleic acid having a base sequence of 10 bases or more continuous selected from a base sequence having 90% or more homology with the base sequence shown in FIG. 1, and a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 51 The virus detection method according to claim 3 or 4, which is a nucleic acid obtained by a nucleic acid amplification treatment using two selected groups as a set of amplification primers, consisting of nucleic acids having a base sequence of 10 bases or more.

[6] The nucleic acid obtained by performing the amplification treatment is a nucleic acid having a nucleotide sequence having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 49, and 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 50 A nucleic acid obtained by a nucleic acid amplification process using two selected nucleic acid groups as a set of amplification primers, and a nucleic acid having 90% or more homology with the nucleic acid having the nucleotide sequence of SEQ ID NO: 51 The virus detection method according to claim 5, wherein

[7] Using a nucleic acid having a base sequence of 10 or more bases selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 51 as a probe, By detecting hybrids with nucleic acids (including their amplification products) in the detection target

The method for detecting a virus according to any one of claims 1 to 6, wherein hepatitis E virus is detected.

[8] Using a nucleic acid having a base sequence of 10 bases or more selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 52 as a probe, the probe and the nucleic acid in the detection target The method for detecting a virus according to any one of claims 1 to 6, wherein hepatitis E virus (GI strain) is detected by detecting a hybrid (including the amplification product).

[9] In the virus detection method, a nucleic acid to be detected is a nucleic acid having a base sequence continuous for 10 bases or more selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 49; A nucleic acid having a base sequence of 10 or more consecutive bases selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 50, and a base having 90% or more homology with the base sequence shown in SEQ ID NO: 52 The method for detecting a virus according to claim 8, which is a nucleic acid obtained by a nucleic acid amplification treatment using two selected groups as a set of amplification primers. .

[10] A nucleic acid having a base sequence of 10 bases or more selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 53 as a probe, and the nucleic acid in the probe and the detection target The method for detecting a virus according to any one of claims 1 to 6, wherein hepatitis E virus (GII strain) is detected by detecting ibrids and ibridis.

[11] In the virus detection method, the nucleic acid to be detected is a nucleic acid having a base sequence continuous for 10 bases or more selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 49; A nucleic acid having a base sequence of 10 or more consecutive bases selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 50, and a base having 90% or more homology with the base sequence shown in SEQ ID NO: 53 The method for detecting a virus according to claim 10, which is a nucleic acid obtained by a nucleic acid amplification treatment using two selected groups as a set of primers for amplification. .

[12] Using a nucleic acid having a base sequence of 10 bases or more selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 54 as a probe, The method for detecting a virus according to any one of claims 1 to 6, wherein hepatitis E virus (GIII strain) is detected by detecting hybridization with a nucleic acid in a detection target.

[13] In the virus detection method, the nucleic acid to be detected is a nucleic acid having a base sequence continuous for 10 bases or more selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 49; A nucleic acid having a base sequence of at least 10 bases selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 50, and a base having 90% or more homology with the base sequence shown in SEQ ID NO: 54 The method for detecting a virus according to claim 12, which is a nucleic acid obtained by a nucleic acid amplification treatment using two selected groups as a pair of amplification primers. .

[14] Using a nucleic acid having a base sequence of 10 or more bases selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 55 as a probe, the probe and the nucleic acid in the detection target The method for detecting a virus according to any one of claims 1 to 6, wherein hepatitis E virus (GI V strain) is detected by detecting the above and ibridiz.

[15] In the virus detection method, a nucleic acid to be detected is a nucleic acid having a base sequence continuous for 10 bases or more selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 49; A nucleic acid having a base sequence of 10 or more bases selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 50, and a base having 90% or more homology with the base sequence shown in SEQ ID NO: 55 The method for detecting a virus according to claim 14, which is a nucleic acid obtained by a nucleic acid amplification treatment using two kinds selected as a set of primers for amplification, wherein the group power selected is a nucleic acid power of a base sequence continuous for 10 bases or more selected from the sequence .

[16] A nucleic acid having a base sequence of 10 or more bases selected from a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 49, and 90% or more homology with the base sequence shown in SEQ ID NO: 50 A hepatitis E virus gene amplification primer set comprising a nucleic acid having a base sequence of 10 bases or more selected from a base sequence having

[17] A nucleic acid having a nucleotide sequence having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 49, and a nucleic acid having a nucleotide sequence having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 50 Primer set for hepatitis B virus gene amplification.

[18] selected from nucleotide sequences having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 51 1 A nucleic acid having a base sequence of 0 bases or more.

[19] selected from nucleotide sequences having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 52 1

A nucleic acid having a base sequence of 0 bases or more.

[20] selected from nucleotide sequences having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 53 1

A nucleic acid having a base sequence of 0 bases or more.

[21] selected from nucleotide sequences having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 54 1

A nucleic acid having a base sequence of 0 bases or more.

[22] selected from nucleotide sequences having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 55 1

A nucleic acid having a base sequence of 0 bases or more.

[23] A kit for detecting hepatitis E virus comprising the following elements:

1) An E-type consisting of a nucleic acid having a nucleotide sequence having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 49, and a nucleic acid having a nucleotide sequence having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 50 Primer set for hepatitis virus gene amplification.

2) a probe nucleic acid having a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 51, and Z or a base sequence having 90% or more homology with the base sequence shown in SEQ ID NOs: 52 to 55 A probe nucleic acid set.