KR101256984B1 - Hepatitis E virus detecting method and kit using reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay - Google Patents

Abstract

The present invention is a hepatitis E virus gene-specific primers and probes for detecting HEV, hepatitis E virus gene detection kit comprising the primers and probes, RT-PCR and ELISA using the primers and probes to perform E A method for detecting a gene of hepatitis virus. According to the kit and the detection method of the present invention, the sensitivity and specificity for HEV can be high, and a large amount of samples can be examined at a time, thereby replacing the conventional hepatitis E virus detection method.

Description

Hepatitis E virus detecting method and kit using reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay}

The present invention is a hepatitis E virus gene-specific primers and probes for detecting HEV, hepatitis E virus gene detection kit comprising the primers and probes, RT-PCR and ELISA using the primers and probes to perform E A method for detecting a gene of hepatitis virus.

Hepatitis E virus (hereafter referred to as HEV) is a 30-34 nm size, envelopeless Caliciviridae family, a positive strand RNA virus (Robert H. Purcell and Suzanne U. Emerson, 1990, Fields). Virology, 3051-3061). The virus is one of the representative viral food poisoning strains, and is mainly infected by food in the fecal-oral route (Hui, Y., Sattar, SA, Murrell, KD, Nip, W., Stanfield, PS (Eds.), 2001 , Hepatitis A and E viruses, vol. 2: Viruses, Parasites, Pathogens, and HACCP, 2nd ed.Marcel Dekker, Inc.), especially genotypes 3 and 4, in humans as well as pigs, wild boars, wild deer, wild mongoose, etc. A novel virus in swine is closely related to (Meng, XJ, Purcell, RH, Halbur, PG, Lehman, JR, Webb, DM, Tsareva, TS, Haynes, JS, Thacker, BJ, Emerson, SU, 1997. the human hepatitis E virus.Proc.Natl.Acad.Sci.USA 94, 98609865 .; Okamoto, H., Takahashi, M., Nishizawa, T., Fukai, K., Muramatsu, U., Yoshikawa, A., Tian-Cheng Li, Tatsuo Miyamora, Naokazu Takeda, 2007, Am. J. Trop. Med. Hyg., 76 (1), 170-172, Detection of hepatitis E virus RNA from the bivalve Yamato -Shijimi ( corbicula japonica ) in Japan) and in case of Philippine Colson et al., J. Infectious Diseases, 2010, 202 (6), 825-834, Pig liver sausage as a source of hepatitis E virus transmission to humans. Has been reported.

The infection caused by the hepatitis E virus is oral, and when infected, there are symptoms such as fatigue, nausea and anorexia, and it causes only acute hepatitis and does not progress chronically, but some of the hepatitis E infections are fatal acute hepatitis. Has been reported to have a high mortality rate, especially in pregnant women. Therefore, the accurate diagnosis of hepatitis by detecting the hepatitis E virus is a very important medical problem.

However, the culturing method for the hepatitis E virus is not particularly known, and since the sensitivity is low to detect in the existing technology, there are many difficulties in research related to the world.

In particular, many studies on gastrointestinal viruses causing viral food poisoning have been conducted to detect pathogens in feces, blood, and other specimens. However, few studies have been conducted on concentrated products, so the standard detection method is inadequate. to be. In addition, genetic testing methods such as polymerase chain reaction (PCR) and reverse trancription polymerase chain reaction (hereinafter referred to as RT-PCR), which are currently used molecular biology research techniques, are not suitable for auditing multiple samples at the same time. It is difficult to detect with a single primer pair and can be detected only by increasing sensitivity through nested RT-PCR. However, the primer has a high cost and takes a long time to detect. In addition, a DNA chip technology for detecting multiple pathogens is being developed, but this also has a high development cost and a high test cost per sample has been pointed out as a disadvantage.

Accordingly, the present inventors have made efforts to overcome the low sensitivity of the RT-PCR technique, which is generally used for detecting viral food poisoning strains, and the disadvantage of being inadequate for testing a large amount of samples. When the detection is performed by combining enzyme immunoassay method (Enzyme Linked Immunosorbent Assay, or ELISA) with RT-PCR technique, it is possible to examine a large amount of samples at high sensitivity and specificity. It was confirmed that the conventional hepatitis E virus detection method can be replaced, the present invention was completed.

It is an object of the present invention to provide primers and probes for HEV detection, specific for hepatitis E virus genes.

Another object of the present invention is to provide a hepatitis E virus gene detection kit comprising the primer and probe.

Another object of the present invention is to provide a method for detecting the gene of hepatitis E virus using RT-PCR and ELISA.

In order to achieve the above object, the present invention provides a primer for detecting hepatitis E virus gene, one or more selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9.

As used herein, the term "primer" refers to a short nucleic acid sequence that can form a base pair with a complementary template with a nucleic acid sequence having a short free 3-terminal hydroxyl group and that serves as a starting point for template strand copying.

The primers can initiate RNA or DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, RNA or DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. The primer of the present invention may have 7 to 50 nucleotide sequences as a HEV gene specific primer.

In addition, the primers may incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis. Modifications can also be made using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", substitution of one or more natural nucleotides into homologues, and modifications between nucleotides, such as uncharged linkers such as methylphosphonate, phosphotriester, (E.g., phosphoramidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. Nucleic acid sequences of the invention can also be modified using a label that can provide a detectable signal directly or indirectly. Examples of labels include radioisotopes, fluorescent molecules, biotin, and the like.

The inventors have developed novel HEV-specific primer sets centered on the most homologous parts through complete genomes of human and porcine HEVs from each genotype GI to GIV through literature studies, and are shown in Table 3. The primer of the present invention preferably consists of any one selected from the base sequences described in SEQ ID NO: 1 to SEQ ID NO: 4 (left primer) and any one selected from the base sequences described in SEQ ID NO: 5 to SEQ ID NO: 9 (right primer) It may be a primer set. Preferably, SEQ ID NO: 2 (HEV-L5612) and SEQ ID NO: 9 (HEV-R6018), SEQ ID NO: 2 and SEQ ID NO: 8 (HEV-R5928), SEQ ID NO: 3 (HEV-L5699), SEQ ID NO: 8, SEQ ID NO: 3 And SEQ ID NO: 9, SEQ ID NO: 4 (HEV-L5909) and SEQ ID NO: 9 primer set.

In the present invention, for detection of HEV RNA in a biological sample, using the RNA sequence of a specific region of HEV contained in the biological sample as a template, after reverse transcription, the product is recovered and amplified using the HEV-specifically designed primer. , The amplified product is hybridized with a specifically designed probe (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 11) in a microplate well, and the reaction product is HEV can be detected by measuring the absorbance indicated by the enzymatic action of the enzyme by binding to the labeled antibody.

Conventional methods used RT-PCR alone, but by using the ELISA in the present invention, it is characterized by high sensitivity and specificity for HEV and can test a large amount of samples at once.

As used herein, the term “amplification” refers to an iterative process in which a nucleic acid is replicated. Polymerase chain reaction, ligase chain reaction, real-time PCR, RT-PCR may be used to recover the sequence of a specific region of HEV, but is not limited thereto. Preferably, PCR may be used. Preferably, RT-PCR may be performed to reverse transcription of RNA, followed by PCR to amplify the nucleic acid. Typically, a reaction mixture containing all of the essential components for PCR (including HEV-specific amplification primers) can be added directly to the reverse transcription reaction mixture.

In the present invention, the term "RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) means reverse transcription polymerase chain reaction, and is one of gene amplification method. Taq DNA polymerase used in PCR is an enzyme that amplifies DNA by template DNA. Since RNA can't be amplified directly from RNA, it is called RT-PCR when RNA is templated and then amplified, and then amplified, so it is an enzyme that synthesizes RNA before PCR. The reverse transcriptase can be used to identify a virus because it can replicate millions of times the viral genetic material, and also prepare primers for specific nucleotide sequences of a specific virus and then use the RT-PCR. Can be used for law.

Detection of the HEV-specific amplification product indicates the presence of HEV RNA in the biological sample. When using gel electrophoresis, HEV-specific amplification products can be identified by their size, as predicted by the position in the HEV RNA of the sequence corresponding to the amplification primers used in the reaction. In the present invention, HEV can be detected using an ELISA for the amplification product. The ELISA is described in detail below.

In the present invention, the conditions suitable for the reverse transcription are shown in Table 1, and the reagents and conditions used in the PCR after the RT process are shown in Table 2.

In another aspect, the present invention provides a hepatitis E virus gene detection kit comprising the HEV specific primer of the present invention.

The kit may further comprise at least one probe selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 11.

In a specific embodiment, the kit may be a kit for detecting HEV genes, which includes essential elements necessary for performing reverse transcriptase and enzyme immunoassay. The kit may include individual primer pairs and probes specific for the HEV gene, in addition to test tubes or other suitable containers, reaction buffers (variable pH and magnesium concentrations), deoxynucleotides (dNTPs), Taq-polymers Enzymes such as lyases and reverse transcriptases, DNAse, RNAse inhibitors DEPC-water, sterile water, instructions and the like may further be included.

The kit may also preferably comprise the necessary elements necessary to perform an ELISA, specifically reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes ( Eg conjugated with an antibody) and its substrate or other substance capable of binding to the antibody, and the like.

A schematic diagram of a kit as one specific example using the RT-PCR ELISA of the present invention is shown in FIG. 3. As shown in FIG. 3, RT-PCT ELISA for detecting the amount of viral genome was performed on a streptavidin-coated microtiter plate with a specific probe bound to digoxigenin (Dig) -labeled replicon and hybridized biotin. Detection by immobilization of the stomach and immobilization of immobilized hybridized amplification products can be carried out using anti-Dig antibodies bound to peroxidase or alkaline phosphatase with a final color developmental stage.

Genes of hepatitis E virus can be detected by performing RT-PCR and ELISA on HEV RNAs derived from biological samples using the kit of the present invention.

As one embodiment of the present invention, the HEV detection experiment by performing RT-PCR ELISA using the HEV-specific primer and probe of the present invention, as described in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 Probe showed excellent sensitivity, SEQ ID NO: 2 (HEV-L5612) and SEQ ID NO: 9 (HEV-R6018), SEQ ID NO: 2 and SEQ ID NO: 8 (HEV-R5928), SEQ ID NO: 3 (HEV-L5699) The primer combinations of SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 9, SEQ ID NO: 4 (HEV-L5909) and SEQ ID NO: 9 exhibited high sensitivity to HEV, especially in the primer combinations of L5699R6018 and L5909R6018. Indicated. In addition, it was confirmed that the conventional nested RT-PCR was positive even in the 081010 sample showing positive in single RT-PCR through the designed primer (see FIGS. 4 and 5). In addition, the results of the sensitivity and specificity test showed that the sensitivity and specificity for HEV was excellent (see FIG. 6).

Thus, when the HEV gene is detected using the primers, probes and kits of the present invention, it is difficult to detect a single primer pair due to its low sensitivity, and it is possible to solve the conventional problem that was possible only by increasing the sensitivity through nested RT-PCR. I can see that

As used herein, the term “biological sample” includes, but is not limited to, a sample such as tissue, cells, whole blood, serum, plasma, saliva, sputum, or manure from which the HEV gene can be obtained.

HEV detectable by the present invention may be HEV serotypes 3 and 4, but is not limited thereto.

In another embodiment, the present invention provides a method for detecting hepatitis E virus gene, which comprises (i) at least one RNA from a biological sample selected from the group consisting of nucleotide sequences represented by SEQ ID NOs: 1 to 9. Performing RT-PCR to generate an amplification product; And (ii) detecting the amplification product via ELISA (Enzyme Linked Immunosorbent Assay), wherein detection of the amplification product indicates the presence of HEV RNA in a biological sample. It provides a method for detecting.

In the present invention, the term "ELISA (Enzyme Linked Immunosorbent Assay)" refers to an enzyme-linked immunosorbent assay, which refers to a method of confirming an antigen-antibody reaction by binding an enzyme to an antibody, and used in the present invention to detect an HEV gene. . This method is simple, inexpensive, capable of large-scale analysis, and has high specificity and sensitivity by covalently attaching enzymes to antibody molecules, which is advantageous for qualitative and quantitative analysis and is widely used clinically. This technique uses an antibody that covalently binds the enzyme without altering the catalysis of the enzyme and the specificity of the antibody. Typical binding enzymes include horseradish peroxidase, alkaline phosphatase and β-galactosidase, all of which catalyze reactions that can be measured in very small amounts due to the color of the reaction product. ELISA includes direct ELISA for detecting antigen, indirect ELISA for detecting antibody, direct sandwich ELISA using another labeled antibody that recognizes the antigen in a complex of antibody and antigen attached to a solid support, antibody and antigen attached to a solid support. Indirect sandwich ELISA using a labeled secondary antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex, but is included without limitation in the present invention.

The primer of step (i) may be a primer set consisting of any one selected from the base sequences described in SEQ ID NO: 1 to SEQ ID NO: 4 and any one selected from the base sequences described in SEQ ID NO: 5 to SEQ ID NO: 9.

In addition, the step (ii) may be performed using a probe selected from at least one group consisting of the nucleotide sequences set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 11. have.

RT-PCR may be used in the amplification step of the detection method, and all of the above-described details may be applied to the detection method as it is.

As one embodiment of the present invention, RT-PCR ELISA was carried out according to the detection method using the HEV-specific primers and probes of the present invention, and as set forth in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 Probe showed excellent sensitivity, SEQ ID NO: 2 (HEV-L5612) and SEQ ID NO: 9 (HEV-R6018), SEQ ID NO: 2 and SEQ ID NO: 8 (HEV-R5928), SEQ ID NO: 3 (HEV-L5699) The primer combinations of SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 9, SEQ ID NO: 4 (HEV-L5909) and SEQ ID NO: 9 exhibited high sensitivity to HEV, especially in the primer combinations of L5699R6018 and L5909R6018. Indicated. In addition, it was confirmed that the conventional nested RT-PCR was positive even in the 081010 sample showing positive in single RT-PCR through the designed primer (see FIGS. 4 and 5). In addition, the results of the sensitivity and specificity test showed that the sensitivity and specificity for HEV was excellent (see FIG. 6).

Through this, the HEV detection method to which the RT-PCR ELISA of the present invention is applied can be examined a plurality of samples at the same time, it can be seen that has a superior sensitivity and specificity compared to the conventional detection method.

As described above, hepatitis E virus gene specific of the present invention, HEV detection primers and probes, and hepatitis E virus gene detection kit comprising the same and RT-PCR and ELISA using the primers and probes together By performing the method for detecting HEV, the sensitivity and specificity for HEV can be high, and a large amount of samples can be examined at a time, thereby replacing the conventional hepatitis E virus detection method.

Figure 1 shows a schematic diagram of the hepatitis E virus gene.
2 is a schematic diagram showing HEV-specific primer and probe positions.
Figure 3 shows a schematic of the kit as one specific example applicable when performing the RT-PCR ELISA. RT-PCT ELISA for detecting the amount of viral genome was performed by attaching a specific probe bound to a digoxigenin (Dig) -labeled replicon and hybridized biotin onto a streptavidin-coated microtiter plate. do. Detection of immobilized hybridized amplification products is carried out using anti-Dig antibodies bound to peroxidase or alkaline phosphatase with a final color developmental stage.
Figure 4 is a specific example of applying the RT-PCR ELISA of the present invention, 6 samples selected from a total of 22 pig fecal samples obtained by using the primer of the present invention RT-PCR products obtained by reading from the plate The results are shown.
FIG. 5 shows the results of treatment of each of the six pig fecal samples by concentration (3 pM, 25 pM) for each primer combination.
6 is a test for sensitivity by diluting the amount of cDNA of HEV by 10-fold dilution from 100ng / ul, and four other human viruses (Hepatitis A virus, Rotavirus, Norovirus GI, Norovirus GII), one animal virus (Feline calicivirus) and The results of the test for specificity are shown.

Hereinafter, the present invention will be described in detail through examples. The following examples are only illustrative of the present invention, and the scope of the present invention is not limited thereby.

Example  One: RNA  detach

RNA isolation method was used a method using Trisol. In a 1.5 ml tube, 0.2 g of pig feces and 1.2 ml of PBS were centrifuged at 1200 × g and 4 ° C. for 30 minutes, and the supernatant was again centrifuged at 16000 × g and 4 ° C. for 10 minutes. After vortexing 500 ul of the supernatant and 500 ul of Trisol LS, the mixture was suspended for 10 minutes. After 10 minutes, 300 ul of chloroform was added and vortexed for 10 minutes. After 10 minutes, centrifugation was performed at 7,500 rpm and 4 ° C. for 15 minutes, and the supernatant was transferred to a new 1.5 ml tube, vortexed with 500 ml of cold isopropanol, and overnight at -20 ° C. The next day, after centrifugation for 30 minutes at 14,000 rpm, 4 ℃, the supernatant was removed, and cold 75% DEPC ETOH 500 ul was added to weak vortex. After centrifugation at 14,000 rpm and 4 ° C. for 30 minutes, the supernatant was removed, dried, and 20 ul of DEPC tertiary sterile distilled water was added and stored at −20 ° C. until use.

Example  2: Reverse transcription  Polymerase chain reaction Revers transcription - 중합체  chain reaction , RT - PCR )

The reagents and conditions used in the reverse transcription process are shown in Table 1 below. In addition, the reagents and conditions used in the PCR after the RT process in the RT-PCR of HEV are shown in Table 2 below.

[Table 1]

[Table 2]

primer  design

In the present invention, HEV-specific primer sets were developed and used based on the most homologous parts through complete genomes of human and porcine HEVs from each genotype GI to GIV through literature studies. It is shown in Table 3 below.

[Table 3]

Probe  design

The probe was designed around a specific nucleotide sequence located in the RT-PCR product, but was produced by labeling with biotin having a length of about 20-30bp.

[Table 4]

The position of the HEV-specific primer and probe of the present invention is shown in Figure 2, the program used for the production of the probe was as follows.

-Clone Manager Professional Suite (Scientific & Educational Software)

Oligo (MedProbe)

Primer Premier (Premier Biosoft)

-NTI software (Invitrogen Inc.)

Example  3: Hybridization  And detection reaction

1% gel agarose and 0.1% Ethidium bromide were added to 1X TAE (Tris-Acetate-EDTA) buffer to make a gel and electrophoresed at 100v for 50 minutes to confirm the amplified RT-PCR product. Liver pre-denaturation. After dispensing 10 ul of denaturing solution into the PCR tube, 5 ul of Dig-labeled PCR-amplified product was dispensed and reacted at room temperature for 10 minutes. 2 ul (1 pmol / ul) of biotinylated type-specific probe suitable for each primer combination was added to each tube, and 188 ul of hybridization buffer was added, followed by reaction at 37 ° C for 10 minutes. The negative control was a DIG-labeled PCR product. Only bays were excluded. After the reaction, 200 ul was transferred to a streptavidin-coated plate well and reacted at 37 ° C. for 60 minutes. The reagents of each well of the plate were removed using a vacuum pump, and then washed four times using 200 ul of washing buffer per well at room temperature. 200 ul of anti-Dig peroxidase-conjugated solution was added to each well and allowed to react for 30 minutes at room temperature (* anti-dioxigenin-POD conjugate (peroxidase conjugated anti-DIG antibody): conjugate buffer = 1: 500). Reagents of each well of the plate were removed using a vacuum pump, and then washed four times using 200 ul of washing buffer per well at room temperature. The reaction was confirmed at 30, 60, and 90 minutes after the dark reaction of 200 ul of ABTS substrate solution at room temperature. Absorbance was measured at 405 nm using a microplate reader (reference 492 nm).

Example  4: RT - PCR ELISA Applied HEV Detection test

For six samples selected from a total of 22 pig fecal samples, either one of the left primer sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4 and the right primer sequence shown in SEQ ID NO: 5 to SEQ ID NO: 9 are set After using the primer set of the present invention to obtain RT-PCR products using the probe of the present invention described in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: The plate was read on a plate and tested for detecting HEV.

The 081021 sample showed a positively positive primer combination through conventional PCR for each primer set and the nested RT-PCR using a reference primer was positive, but the single negative RT-PCR was designed through the designed primer. Six samples were selected, including the 081010 sample shown. Then, the signal concentrations were measured for 10 minutes up to 90 minutes with primer concentrations of 3pM and 25pM, and the probe concentration, time, and measurement were selected for the positive determination, and the sensitivity of each probe for each primer combination was compared. Probes with high signal were selected.

As a result, the probe described in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 showed excellent sensitivity. In addition, as shown in Figures 4 and 5, RT-PCR ELISA using the HEV-specific primers and probes of the present invention, showed a high sensitivity for HEV for each primer combination, in particular L5699R6018 and L5909R6018 Excellent sensitivity was shown in the primer combination of. In addition, the conventional nested RT-PCR showed a positive, but it was confirmed that even in the 081010 sample showing a certain negative in a single RT-PCR through the designed primer. As a result, when using the method of the present invention, multiple samples can be tested simultaneously, and the sensitivity is low, so it is difficult to detect with a single primer pair, and it is possible to replace the conventional test method which was possible to increase the sensitivity through nested RT-PCR. It can be seen that.

Example  5: RT - PCR ELISA Applied HEV Sensitivity and specificity of detection methods

Sensitivity was tested by diluting the amount of cDNA of HEV in 10-fold steps from 100 ng / ul and testing specificity with 4 other human viruses (Hepatitis A virus, Rotavirus, Norovirus GI, Norovirus GII) and 1 animal virus (Feline calicivirus). Was carried out. As a result, as shown in Figure 6, it showed an excellent effect in both sensitivity and specificity.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Hepatitis E virus detecting method and kit using reverse          transcription-polymerase chain reaction enzyme-linked          immunosorbent assay <160> 11 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV-L5227 <400> 1 gttgctcttc gtgcttctgc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV-L5612 <400> 2 aatttgtcta cgtccccgct 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV-L5699 <400> 3 cttcaggatg gcaccaacac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV-L5909 <400> 4 gttcagcccg gtatagcctc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV-R5632 <400> 5 aagcggggac gtagacaaat 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV-R5718 <400> 6 gtgttggtgc catcctgaag 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV-R5818 <400> 7 atagccacca acagcattcg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV-R5928 <400> 8 gaggctatac cgggctgaac 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV-R6018 <400> 9 gtagcctcct cctcagccac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV-5359P <400> 10 gggctgagaa tcaaccctgt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV-5952P <400> 11 gccttcacta ccgcaatcaa 20

Claims (9)

A primer set for detection of hepatitis E virus gene consisting of the nucleotide sequences set forth in SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 9. delete delete Hepatitis E virus gene detection kit comprising the primer set of claim 1. The kit of claim 4, wherein the kit further comprises at least one probe selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 7. 6. (i) Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a primer for detecting hepatitis E virus gene consisting of a nucleotide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 9 Performing an amplification product; And
(ii) detecting the amplification product via an ELISA (Enzyme Linked Immunosorbent Assay); wherein the detection of the amplification product indicates the presence of HEV RNA in a biological sample. How to. delete 7. The method of claim 6, wherein step (ii) is performed using a probe selected from at least one group consisting of the nucleotide sequences set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 7. delete

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