KR101763607B1 - Hepatitis A virus standardized positive control gene and RNA transcripts transcripted therefrom - Google Patents

Abstract

The present invention relates to a hepatitis A virus standard positive control gene, a recombinant vector comprising the same, an RNA transcript transcribed from the gene, And a detection kit comprising the RNA transcript. The hepatitis A virus standard positive control gene according to the present invention and the RNA transcript transcribed therefrom are different from the hepatitis A virus PCR product detected in an environmental sample and are different in size from each other and false positives due to cross contamination of environmental samples positive and false-negative, respectively. Thus, it can be used accurately and quickly for the detection of hepatitis A virus.

Description

A hepatitis A virus standard positive control gene and RNA transcripts transcribed therefrom (Hepatitis A virus standardized positive control gene and RNA transcripts transcripted therefrom)

The present invention relates to a hepatitis A virus standard positive control gene, a recombinant vector comprising the same, an RNA transcript transcribed from the gene, And a detection kit comprising the RNA transcript.

Hepatitis A virus is predominantly transmitted through contaminated food or water, causing hepatitis and liver failure, and is closely related to the health and hygiene and socioeconomic living standards of the area (Fields virology, David M Knipe, Peter M. Howley, Fourth edition, 2001). Initially, it was classified as enterovirus type 72, but due to differences in biochemical and physical properties, it was isolated from enterovirus and transferred to a new genome called Hepatovirus of Picornaviridae family . The hepatitis A virus is divided into various strains according to the area and the time point at which the virus is isolated. The most representative strains were HM175 (Australia, 1976), CR326 (Costa Rica, 1960), MS-1 (New York, 1964), SD11 (Califonia, 1974) (Owl monkey, Panama, 1980) and AGM27 (African green monkey, Panama, 1980), which are isolated from animals, Russia, 1985). Although these strains are basically classified according to their growth characteristics, most of them are classified based on genetic sequence. Hepatitis A virus does not have an envelope but is stable in the intestinal tract of bile and various conditions and is viable in many environments such as seawater, wastewater, soil and oyster. This can be attributed to the physical and chemical resistance of various environments of hepatitis A virus.

Real-time gene amplification (Realtime RT-PCR) and conventional gene amplification (Conventional RT-PCR) are commonly used to detect hepatitis A virus. However, in the case of the general gene amplification method, there is a difficulty in using a virus recovered by culturing in an animal cell as a PCR positive control used for testing the amplification test procedure. That is, a cell culture system for continuous use in a laboratory or a laboratory to be tested can be used as a positive control. These problems can cause problems in the continued supply of PCR positive controls. In addition, when hepatitis A virus obtained through cell culture is used, there is a risk of false positives due to crossing risk caused by carry over during the PCR process, and the result of false positives In order to confirm this, it is difficult to confirm the contamination by comparing the base sequence analysis between the actual analysis sample and the positive control group. In order to improve the real limitations and problems of the positive control group of hepatitis A virus, the development of a standard positive control group in which the sample and the PCR size are distinguished from each other by applying the same sample to the actual assay mixture and the amplification and reagent mixing conditions It is necessary.

Accordingly, the present inventors have continued to develop a positive control for hepatitis A virus, and as a result, have found that bidirectional primers for amplifying the capsid gene of hepatitis A virus; And the hepatitis A virus genotype 41 (enteric adenovirus) gene inserted into the gene of the hepatitis A virus standard positive control gene and the RNA transcripts transcribed from the virus when the detection of hepatitis A virus It can be performed accurately and quickly, thereby completing the present invention.

An object of the present invention is to provide a forward primer for amplifying a capsid gene of hepatitis A virus; A gene for hepatitis A virus; Enteric adenovirus type 41 gene; And a reverse primer that amplifies a capsid gene of hepatitis A virus, and a positive control gene for hepatitis A virus.

It is also an object of the present invention to provide a recombinant vector comprising a hepatitis A virus standard positive control gene.

It is also an object of the present invention to provide a hepatitis A virus standard positive control RNA transcript transcribed from a hepatitis A virus standard positive control gene.

It is also an object of the present invention to provide a kit for detecting hepatitis A virus comprising a hepatitis A virus standard positive control RNA transcript.

In order to solve the above-mentioned problems, the present invention provides a primer for amplifying a capsid gene of hepatitis A virus; A gene for hepatitis A virus; A superficial adenovirus type 41 gene; And a reverse primer for amplifying the capsid gene of hepatitis A virus. The present invention also provides a standard positive control gene for hepatitis A virus.

The present invention also provides a recombinant vector comprising a hepatitis A virus standard positive control gene.

The present invention also provides a standard positive control RNA transcript of hepatitis A virus transcribed from a hepatitis A virus standard positive control gene.

The present invention also provides a kit for detecting hepatitis A virus comprising a hepatitis A virus standard positive control RNA transcript.

The hepatitis A virus standard positive control gene according to the present invention and the RNA transcript transcribed therefrom are different from the hepatitis A virus PCR product detected in an environmental sample and are different in size from each other and false positives due to cross contamination of environmental samples positive and false-negative, respectively. Thus, it can be used accurately and quickly for the detection of hepatitis A virus.

FIG. 1 shows the results of RT-PCR of the hepatitis A virus detected in FRhK- 4 cells infected with hepatitis A virus (HM-175) as a sample using a primer for VP3 / VP1 site detection and electrophoresis (Lane 1: PCR size marker marker, lane 2: VP3 / VP1 site).
FIG. 2 shows the results of RT-PCR of the hepatitis A virus detected in the FRhK- 4 cell infected with the hepatitis A virus (HM-175) as a sample using a primer for detecting the VP1 / P2A region and confirmed by electrophoresis (Lane 1: PCR size mark marker, lane 2: VP1 / P2A site).
3 is a forward primer for amplifying the capsid gene of hepatitis virus; A gene for hepatitis A virus; Enteric adenovirus type 41 gene; And a reverse primer that amplifies the capsid gene of hepatitis A virus into a vector.
FIG. 4 shows electrophoretic analysis of recombinant plasmid extracted and transformed with transformed E. coli cells DH5α (lane 1: PCR size mark marker, lane 2: recombinant plasmid band extracted and purified from DH5a ).
FIG. 5 shows the result of electrophoresis of the result of treatment of the recombinant plasmid with restriction enzyme ECoR I (lane 1: PCR size marker marker, lane 2: recombinant vector treated with restriction enzyme ECoRI).
FIG. 6 is a diagram showing the result of electrophoresis after treating the plasmid with restriction enzyme Spe I (lane 1: PCR size mark marker, lane 2: band treated with plasmid with SpeI).
FIG. 7 shows the results of immunohistochemical staining for hepatitis A virus detected in FRhK-4 cells infected with hepatitis A virus (HM-175), a standard positive control for hepatitis A virus; (Lane 1: PCR size marker marker, lane 2: VP3 / VP1 site), and the primer for detection of VP3 / VP1 region Lane 3: VP3 / VP1 site PCR positive control).
FIG. 8 shows the results of immunohistochemical staining for hepatitis A virus detected in FRhK-4 cells infected with hepatitis A virus (HM-175), a standard positive control for hepatitis A virus; (Lane 1: PCR size marker marker, lane 2: VP1 / P2A site, lane 3: VP1), and VP1 / P2A site primer / P2A site PCR positive control).

The present invention relates to a forward primer for amplifying a capsid gene of hepatitis A virus; A gene for hepatitis A virus; A superficial adenovirus type 41 gene; And a reverse primer for amplifying the capsid gene of hepatitis A virus. The present invention also provides a standard positive control gene for hepatitis A virus.

In the present invention, "standard positive control group" means a positive control group used for standardization in detecting hepatitis A virus in a sample using a molecular biological method.

Generally, a positive control group will be used for objective and reliable detection of hepatitis A virus. However, the positive control group has a problem in that it is difficult to use the recovered virus after cultivation in animal cells and there is a problem in continuous supply. Currently, there is no standard positive control required for detection of hepatitis A virus, and a standardized positive control Is urgently needed.

In the present invention, a "primer" refers to a single strand oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primer may depend on the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target. The oligonucleotide used as the primer may also comprise a nucleotide analogue such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid or may be intercalated agent.

In the present invention, the hepatitis A virus standard positive control gene can be artificially synthesized by using a nucleic acid synthesizer or the like with reference to the base sequence of the gene of interest, or a sequence complementary to both ends of a desired leptin gene using leptin genomic DNA as a template Lt; RTI ID = 0.0 > oligonucleotide < / RTI > On the other hand, the leptin gene of the present invention can exist in various base sequences due to codon degeneracy, all of which are included in the scope of the present invention. In addition, variants of the hepatitis A virus standard positive control gene are included in the scope of the present invention. Specifically, a base having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the nucleotide sequence of the hepatitis A virus standard positive control gene Sequence. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The hepatitis A virus standard positive control gene may be introduced into cells by known methods in the art, for example, naked DNA in the form of a vector (Wolff et al. Science, 1990: Wolff et al. J Cell Sci. 59, 1992), liposomes, cationic polymers, and the like.

The capsid gene of the hepatitis A virus may be a VP3 / VP1 or VP1 / P2A region, and the forward primer may be a nucleotide sequence of SEQ ID NOS: 1, 3, 5 and 7. The hepatitis A virus gene may be a nucleotide sequence of SEQ ID NO: 9, and the adenovirus type 41 gene may be a nucleotide sequence of SEQ ID NO: 10.

In one embodiment of the present invention, a forward primer that amplifies a capsid gene of hepatitis A virus; A gene for hepatitis A virus; A superficial adenovirus type 41 gene; And a reverse primer that amplifies the capsid gene of hepatitis A virus were synthesized and cloned into pGEM T-easy vector. Thereafter, the recombinant vector was transcribed in vitro using T7 RNA polymerase to prepare a standard positive control RNA transcript of hepatitis A virus.

The present invention also provides a recombinant vector comprising a hepatitis A virus standard positive control gene.

As used herein, the term "vector" refers to an expression vector capable of expressing a protein of interest in a suitable host cell, which gene construct comprises an essential regulatory element operably linked to the expression of the gene insert.

The vector of the present invention may include a signal sequence or a leader sequence for membrane targeting or secretion in addition to an expression regulatory element such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, an enhancer, and the like. The promoter of the vector may be constitutive or inducible. In addition, the expression vector includes a selectable marker for selecting a host cell containing the vector, and includes a replication origin in the case of a replicable expression vector. The vector may be self-replicating or integrated into the host DNA.

The present invention also provides a standard positive control RNA transcript of hepatitis A virus transcribed from a hepatitis A virus standard positive control gene.

The RNA transcript may be transcribed in vitro using a T7 RNA polymerase from a recombinant vector having the cleavage map of FIG.

In the present invention, the RNA transcript can be used as a standard positive control group for detecting the detection of hepatitis A virus upon detection of hepatitis A virus from a sample. That is, the standard positive control RNA transcript of the present invention is used as a standard positive control to confirm whether the detection of hepatitis A virus is properly performed in the sample by carrying out reverse transcription polymerization and PCR reaction together with the sample.

The present invention also provides a kit for detecting hepatitis A virus comprising a hepatitis A virus standard positive control RNA transcript.

The diagnostic kit may be manufactured to include a reaction solution according to need, which can be easily performed by applying known techniques. The kit of the present invention may further comprise reagents necessary for the diagnosis of hepatitis A virus, and the reagents may be, for example, buffers and the like. In addition, the kit may include a brochure. The manual is a printed document that explains how to use the kit, for example, how to make the buffer, the reaction conditions presented, and so on. The brochure includes instructions on the surface of the package including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention as defined by the appended claims. It will be obvious to you.

Example 1 Detection of VP3 / VP1 or VP1 / P2A Region of Capsid Gene of Hepatitis A Virus

1-1. RNA isolation of hepatitis A virus

In order to obtain the nucleotide sequence of hepatitis A virus, isolation of hepatitis A virus RNA was performed as follows.

More specifically, a virus RNA kit (QIAamp ^® Viral RNA Mini; QIAGEN, Germany) was used to isolate hepatitis A virus RNA. The samples used were FRhK -4 cells infected with hepatitis A virus (HM-175). 280 μl of the above-mentioned sample centrifuged at 14,000 rpm for 3 minutes and 1,120 μl of AVL buffer (including carrier RNA) were placed in a 15 ml tube and pulse-vortexed for 15 seconds, then allowed to stand at room temperature for 10 minutes, Down. 1,120 μl of ethanol (96-100%) was added to the tube, mixed for 15 seconds, and then spun down again. 630 μl of the mixed sample was put into a spin column (QIAamp Mini spin column) (containing 2 ml tube) and centrifuged at 6,000 × g for 1 minute to effect RNA adsorption. For the first wash, the filtered liquid and tube were discarded and the spin column was transferred to a new 2 ml tube, followed by 500 ul AW1 wash buffer and centrifugation at 6,000 xg for 3 minutes. For the second wash, the first washed spin column was transferred to a new 2 ml tube, then 500 μl of AW2 wash buffer was added and centrifuged at full speed for 1 minute. The second washed spin column was loaded in a 1.5 ml tube, and 60 쨉 l of AVE elution buffer was added to the column. After being left for 1 minute, RNA was isolated from Hepatitis A virus by centrifugation at 6,000 xg for 1 minute. When not used immediately, the separated RNA was stored at -70 ° C.

1-2. Reverse transcription polymerase chain reaction (RT-PCR) and semi-nested PCR

1-step RT-PCR and semi-nested PCR were performed to amplify the hepatitis A virus capsid gene VP3 / VP1 or VP1 / P2A in hepatitis A virus isolated in Example 1-1 .

More specifically, in order to perform 1-step RT-PCR, 25 μl of 1-step PCR Reddy mix (Verso 1-step RT-PCR Reddy mix kit; Thermo scientific) 5 μl of Hepatitis A virus RNA isolated, 2 μl each of forward and reverse primers at 20 pmol concentration as shown in Table 1 (VP3 / VP1 amplification primers shown in SEQ ID NOs: 1 and 2, SEQ ID NOs: 5 and 6 1 μl of reverse transcriptase and 15 μl of distilled water were added to a final volume of 50 μl, and 1-step RT-PCR was performed under the conditions shown in Table 2 below.

In order to perform semi-nested PCR, 5 μl of the template cDNA obtained by the above RT-PCR reaction, 20 pmol of the forward and reverse primers (VP3 / VP1 shown in SEQ ID NO: 3 and 4, 5 μl each of dNTPs, 5 μl of 10 × buffer solution, 1 μl of Taq DNA polymerase and 31 μl of distilled water were added to a final volume of 50 μl, Semi-nested PCR was performed.

Amplified region primer Primer base sequence (5 '- > 3') SEQ ID NO: direction VP3 /
VP1 HAV1
HAV2
HAV3
HAV4 GCT CCT CTT TAT CAT GCT ATG GAT
CAG GAA ATG TCT CAG GTA CTT TCT
ATG TTA CTA CAC AAG TTG GAG AT
GAT CCT CAA TTG TTG TGA TAG CT SEQ ID NO: 1 Forward SEQ ID NO: 2 Reverse SEQ ID NO: 3 Forward SEQ ID NO: 4 Reverse VP1 /
P2A BR5
BR9
RJ3
BR6 TTG TCT GTC ACA GAA CAA TCA G
AGT CAC ACC TCT CCA GGA AAA CTT
TCC CAG AGC TCC ATT GAA
AGG AGG TGG AAG CAC TTC ATT TGA SEQ ID NO: 5 Forward SEQ ID NO: 6 Reverse SEQ ID NO: 7 Forward SEQ ID NO: 8 Reverse

Type of reaction Condition Number of repetitive reactions cDNA synthesis
Initial denaturation 48 ° C 40 min
95 ° C 4 min 1 cycle denaturalization
Combination
expansion 95 ° C 30 sec 30 cycles 55 ° C 30 sec 72 ° C 30 sec Final height 72 ° C 7 min 1 cycle keep 4 ℃ 8 ∞

Type of reaction Condition Number of repetitive reactions Initial denaturation 95 ° C 4 min 1 cycle denaturalization
Combination
expansion 95 ° C 30 sec 30 cycles 55 ° C 30 sec 72 ° C 30 sec Final height 72 ° C 7 min 1 cycle keep 4 ℃ 8  ∞

1-3. Electrophoresis analysis

To confirm the hepatitis A virus capsid gene VP3 / VP1 or VP1 / P2A region amplified in Example 1-2, electrophoresis and sequence analysis were performed as follows.

More specifically, to perform electrophoresis, Loading STAR (DyneBioinc, korea) and PCR products amplified in Example 1-2 were mixed in a volume ratio of 1: 5, and 2% agarose gel was added to 0.5 x TAE buffer , And the bands were confirmed. The results are shown in Fig. 1 and Fig.

As shown in Fig. 1, it was confirmed that the PCR product of the hepatitis A virus capsid gene VP3 / VP1 site was 186 bp.

As shown in Fig. 2, it was confirmed that the PCR product of the hepatitis A virus capsid gene VP1 / P2A region was 234 bp.

1-4. Sequencing

PCR of the hepatitis A virus hepatitis virus capsid VP3 / VP1 or VP1 / P2A region confirmed by performing the above-mentioned 1-3 electrophoresis in order to finally determine the hepatitis A virus gene group (HM-175 genogroup) by analyzing the gene base sequence The base sequence was analyzed using the product.

More specifically, the PCR products of the hepatitis A virus capsid VP3 / VP1 or VP1 / P2A site amplification genes were purified using a PCR purification kit (Bioneer, Korea). The purified sample was used as a detection primer (primer represented by SEQ ID NO: 3 and 4, VP1 / P2A site amplified gene in the case of VP3 / VP1 site amplification PCR product, primer represented by SEQ ID NO: 7 and 8) And the nucleotide sequence was analyzed. (3500xL Genetic Analyzer, ABI), and the nucleotide sequence determined by DNA sequencing was compared with that of the hepatitis A virus gene database (NCBI GeneBank) and the amplified hepatitis A virus capsid VP3 / VP1 or The VP1 / P2A site was confirmed to be hepatitis A virus.

The results of the electrophoresis of the above-mentioned Example 1-3 on the finally confirmed VP3 / VP1 or VP1 / P2A regions of the hepatitis A virus capsid gene were compared with the results of the hepatitis A virus standard Detection efficiency of positive control RNA transcripts; And size size comparisons after electrophoresis.

Example 2 Secretion of adenovirus type 41 (Enteric Adeno 41) gene as an external gene and hepatitis A virus standard positive control Gene synthesis

The following experiment was carried out in order to obtain an exogenous adenovirus type 41 gene and to prepare a standard positive control gene for hepatitis A virus, and to purify the cloned, transformed and plasmid using the same.

More specifically, in order to prepare a standard positive control gene for hepatitis A virus, the gene of the external adenovirus 41 was searched (NCBI Genbank no. DQ315364) to obtain the nucleotide sequence shown in SEQ ID NO: 10.

Also, in order to prepare a standard positive control gene for hepatitis A virus, a forward primer (SEQ ID NOS: 1, 3, 5 and 7) amplifying the capsid gene of hepatitis A virus by a gene synthesis company (Bioneer); The gene for hepatitis A virus (SEQ ID NO: 9); A superficial adenovirus type 41 gene (SEQ ID NO: 10); And reverse primers (SEQ ID NOS: 2, 4, 6 and 8) for amplifying the capsid gene of hepatitis A virus were synthesized. The synthesized gene was cloned into a pGEM T-easy plasmid vector, and the vector cleavage map is shown in Fig.

Then, to transform, 1 μl of the synthesized plasmid and 50 μl of E. coli cells in a 1.5 ml tube of DH5 α were added, and 1 μl of the synthesized plasmid was inserted into ice for 20 minutes to induce transformation. After 20 minutes, it was subjected to heat-shock treatment in a constant temperature water bath at 42 ° C for 60 to 90 seconds, and then left in ice for 3 minutes. To screen for ampicillin-resistant transformants, add 10 ml of LB broth containing ampicillin, pipet and mix well and incubate for 16 hours at 37 ° C in a shaking incubator. Lt; / RTI >

Next, Plasmid miniprep kit (Solgent, Korea) was used to purify the plasmid. The culture was transferred to a 1.5 ml tube, centrifuged at 13,000 rpm for 1 minute, and the supernatant in the tube was discarded. To resuspend the pellet in the tube, 250 μl of RNase-treated SP1 buffer was added to the remaining pellet and vortexed. For lysis of the mixed samples, 250 μl of SP2 buffer was added, inverted for 4 to 6 times, and left at room temperature for 2 minutes. In order to neutralize the above-mentioned neutralized sample, 350 μl of SGP3 buffer was added, and the sample was flipped 4 to 6 times, followed by centrifugation at 10,000 rpm for 10 minutes. The lysate was carefully transferred except for the residues present in the tube of the centrifuged sample, carefully placed in a collection tube-coupled column, centrifuged again at 10,000 rpm for 30 seconds Respectively. The residues passed through the column were discarded and the collection tube was again joined to the column. 750 μl of 100% ethanol-added wash buffer (wash buffer) was added to the column and centrifuged at 12,000 rpm for 30 seconds. The residue passed through the column was discarded and the collection tube was again combined with the column and washed once again in the same manner as described above. Centrifuge for 3 minutes at 12,000 rpm for drying of the washed column, transfer the column to a new 1.5 ml tube, add 30 μl of elution buffer (elution buffer) for 2 minutes, Minute centrifugation. The purified plasmid was confirmed by electrophoresis on 2% agarose gel. The results are shown in Fig.

As shown in Fig. 4, it was confirmed that the plasmid extracted and purified from DH5? Was about 3536 bp.

 For the restriction enzyme treatment of the plasmid, 1 μl (10 μl) of restriction enzyme EcoRI (TaKaRa Biotechnology), 1 μl of 10 × buffer, 5 μl of plasmid DNA and 3 μl of distilled water were placed in the tube. The tube was cut for 1 < RTI ID = 0.0 > h < / RTI > When the reaction was completed, the inserted DNA was confirmed by electrophoresis on 2% agarose gel. The results are shown in Fig.

As shown in Fig. 5, it was confirmed that the recombinant plasmid treated with ECoRI restriction enzyme was about 538 bp.

Example 3. Preparation of RNA transcript

In order to prepare an RNA transcript transcribed from a standard positive control gene of hepatitis A virus, the following experiment was conducted.

More specifically, 1 μl of SpeI (10 U) (TaKaRa Biotechnology), 1 μl of 10 × buffer, 5 μl of plasmid DNA, and 1 μl of a 10 × buffer were mixed with restriction enzymes to make linear DNA for the production of in vitro RNA transcripts. 3 μl of distilled water was placed in a 1.5 ml tube and allowed to react for 1½ hours at 37 ° C in an incubator. When the reaction was completed, DNA was confirmed by electrophoresis on 2% agarose gel. The results are shown in Fig.

As shown in Fig. 6, it was confirmed that the plasmid treated with SpeI restriction enzyme was about 3536 bp.

Next, in order to synthesize a large amount of ssRNA by in vitro transcription reaction using T7 RNA polymerase, T7 transcription was performed using plasmid DNA treated with the restriction enzyme having the T7 promoter sequence as a template, Respectively. More specifically, RiboMAX Large Scale RNA Production Systems-T7 kit from Promega, USA was used. Add 20 μl of T7 5 × buffer and 30 μl of rNTPs (25 mM ATP, CTP, GTP, UTP), 20 μl of linear DNA template (7.48 μg) and 30 μl of distilled water to a total volume of 100 μl. And reacted for 4 hours. 10 μl of RQ1 RNAase-Free DNAase (Promega) was added to the reaction solution and reacted at 37 ° C. for 15 minutes in an incubator.

In order to purify the RNA, 100 μl of the above-described T7 transcript and 100 μl of 25: 24: 1 phenol: chloroform: isoamyl (sigma) were mixed in a 1.5 ml tube And then centrifuged at 14,000 rpm for 2 minutes. The supernatant was taken out from the centrifuged tube and placed in a 1.5 ml tube. 24: 1 = chloroform: isoamyl (sigma) was added in the same amount as the supernatant, and the mixture was centrifuged at 14,000 rpm for 2 minutes. The supernatant was taken from the centrifuged tube and 3% sodium acetate (Promega) was added in an amount of 0.1% of the supernatant volume, isopropanol was added to the supernatant in the same amount, and the mixture was reacted on ice for 2 to 5 minutes. The reaction solution was centrifuged at 14,000 rpm for 10 minutes. The supernatant was discarded, and 1 ml of cold 70% ethanol was added thereto. The reaction mixture was flipped and centrifuged at 14,000 rpm for 10 minutes. The supernatant was removed and dried at room temperature for 20 minutes. For elution of the dried sample, 30 μl of distilled water was added and the mixture was allowed to stand at room temperature for 2 minutes. RNA was then stored at -80 ° C.

Example 4. Confirmation of detection efficiency of standard positive control gene for hepatitis A virus prepared for environmental sample

Detection efficiency of the VP3 / VP1 or VP1 / P2A region of the capsid gene of the hepatitis A virus of the RNA transcript prepared in Example 3; And the sizes of the bands and the size of the electrophoresis performed in Example 1-3, the following experiment was performed.

More specifically, in order to perform 1-step RT-PCR, 25 μl of 1-step PCR Reddy mix (Verso 1-step RT-PCR Reddy mix kit; Thermo scientific) , The Hepatitis A virus standard positive control RNA transcript obtained in Example 1, 2 [mu] l each of the 20 pmol concentration forward and reverse primers shown in Table 1 (primers for amplifying VP3 / VP1 represented by SEQ ID NOs: 1 and 2; 6 primer for amplification of VP1 / P2A), 1 μl of reverse transcriptase and 15 μl of distilled water were added to a final volume of 50 μl, and 1-step RT-PCR was performed under the conditions described in Table 2 above.

In order to carry out the semi-nested PCR, 5 μl of the template cDNA obtained by the above RT-PCR reaction, 20 pmol of forward and reverse primers (VP3 / VP1 shown in SEQ ID NO: 3 and 4 5 μl each of dNTPs, 5 μl of 10 × buffer, 1 μl of Taq DNA polymerase and 31 μl of distilled water were added to a final volume of 50 μl, Semi-nested PCR was performed under the conditions described in 3.

Further, electrophoresis was performed to confirm the PCR products. More specifically, loading STAR (DyneBioinc, korea) and PCR products were mixed in a volume ratio of 1: 5 and loaded with 0.5% TAE buffer using 2% agarose gel to confirm bands. The results are shown in Fig. 7 and Fig.

As shown in FIG. 7, it was confirmed that the standard positive control group, VP3 / VP1 site amplification PCR product, was 473 bp, and the nucleotide sequence thereof was shown in SEQ ID NO: 11. Thus, it was confirmed that the size of the amplified gene of VP3 / VP1 region (186 bp) was different from that shown in FIG.

As shown in FIG. 8, it was confirmed that the standard positive control group, VP1 / P2A site amplification PCR product, was 381 bp, and the nucleotide sequence thereof was represented by SEQ ID NO: 12. As a result, it was confirmed that the size of the VP1 / P2A region amplified gene shown in FIG. 2 was different from that of (234 bp).

Therefore, when the hepatitis A virus standard positive control gene of the present invention and the RNA transcript transcribed therefrom are used, it can be used as a standard positive control for the VP3 / VP1 or VP1 / P2A region which is a capsid gene of hepatitis A virus , And that it is useful and convenient to use.

<110> KOREA FOOD & DRUG ADMINISTRATION <120> Hepatitis A virus standardized positive control and RNA          transcripts transcripted therefrom <130> 1-1 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer of HAV1 for VP3 / VP1 <400> 1 gctcctcttt atcatgctat ggat 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer of HAV2 for VP3 / VP1 <400> 2 caggaaatgt ctcaggtact ttct 24 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer of HAV3 for VP3 / VP1 <400> 3 atgttactac acaagttgga gat 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer of HAV4 for VP3 / VP1 <400> 4 gatcctcaat tgttgtgata gct 23 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer of BR5 for VP1 / VP2A <400> 5 ttgtctgtca cagaacaatc ag 22 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer of BR9 for VP1 / VP2A <400> 6 agtcacacct ctccaggaaa actt 24 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer of RJ3 for VP1 / VP2A <400> 7 tcccagagct ccattgaa 18 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer of BR6 for VP1 / VP2A <400> 8 aggaggtgga agcacttcat ttga 24 <210> 9 <211> 140 <212> DNA <213> HAV original sequence <400> 9 gattctggag gtttttcaac aacagtttct acagaacaga atgttccaga tccccaagtt 60 ggtataacaa ccatgaaaga tttgaaagga aaagctaaca gagggaaaat ggatgtttca 120 ggagtacaag cacctgtggg 140 <210> 10 <211> 199 <212> DNA <213> Enteric Adenovirus <400> 10 tggccacccc ctcgatgatg ccgcaatggt cttacatgca catcgccggg caggacgcct 60 cggagtatct gagcccgggc ctggtgcaat ttgcccgcgc caccgatacg tacttcagcc 120 tggggaacaa gttcagaaat cccactgtgg ctccgaccca cgatgtaacc acagacaggt 180 cacagcgact gacgctgcg 199 <210> 11 <211> 473 <212> DNA <213> Artificial Sequence <220> VP3 / VP1 amplification gene synthesis sequence <400> 11 atgttactac acaagttgga gatttgtctg tcacagaaca atcagtccca gagctccatt 60 gaatggccac cccctcgatg atgccgcaat ggtcttacat gcacatcgcc gggcaggacg 120 cctcggagta tctgagcccg ggcctggtgc aatttgcccg cgccaccgat acgtacttca 180 gcctggggaa caagttcaga aatcccactg tggctccgac ccacgatgta accacagaca 240 ggtcacagcg actgacgctg cggattctgg aggtttttca acaacagttt ctacagaaca 300 gaatgttcca gatccccaag ttggtataac aaccatgaaa gatttgaaag gaaaagctaa 360 cagagggaaa atggatgttt caggagtaca agcacctgtg ggtcaaatga agtgcttcca 420 cctcctaagt tttcctggag aggtgtgact agctatcaca acaattgagg atc 473 <210> 12 <211> 381 <212> DNA <213> Artificial Sequence <220> VP1 / P2A amplification gene synthesis sequence <400> 12 tcccagagct ccattgaatg gccaccccct cgatgatgcc gcaatggtct tacatgcaca 60 tcgccgggca ggacgcctcg gagtatctga gcccgggcct ggtgcaattt gcccgcgcca 120 ccgatacgta cttcagcctg gggaacaagt tcagaaatcc cactgtggct ccgacccacg 180 atgtaaccac agacaggtca cagcgactga cgctgcggat tctggaggtt tttcaacaac 240 agtttctaca gaacagaatg ttccagatcc ccaagttggt ataacaacca tgaaagattt 300 gaaaggaaaa gctaacagag ggaaaatgga tgtttcagga gtacaagcac ctgtgggtca 360 aatgaagtgc ttccacctcc t 381

Claims (11)

A forward primer amplifying the VP3 / VP1 capsid gene of hepatitis A virus; A forward primer that amplifies the VP1 / P2A capsid gene of hepatitis A virus; A gene of hepatitis A virus represented by SEQ ID NO: 9; An enteric adenovirus type 41 gene represented by SEQ ID NO: 10; A reverse primer amplifying the VP3 / VP1 capsid gene of hepatitis A virus; And a reverse primer that amplifies the VP1 / P2A capsid gene of hepatitis A virus, and a positive control gene for hepatitis A virus. The method according to claim 1,
Wherein the forward primer for amplifying the VP3 / VP1 capsid gene of the hepatitis A virus is a forward primer represented by SEQ. ID. NO. 1 and a forward primer set represented by SEQ. ID. NO. 3, which is a standard positive control gene for hepatitis A virus. The method according to claim 1,
Wherein the forward primer for amplifying the VP1 / P2A capsid gene of the hepatitis A virus is a forward primer represented by SEQ ID NO: 5 and a forward primer set represented by SEQ ID NO: 7, and a standard positive control gene for hepatitis A virus. The method according to claim 1,
Wherein the reverse primer for amplifying the VP3 / VP1 capsid gene of hepatitis A virus is a reverse primer set forth in SEQ ID NO: 2 and a reverse primer set set forth in SEQ ID NO: 4, and a standard positive control gene for hepatitis A virus. The method according to claim 1,
Wherein the reverse primer amplifying the VP1 / P2A capsid gene of hepatitis A virus is a reverse primer set forth in SEQ ID NO: 6 and the reverse primer set set forth in SEQ ID NO: 8, and a standard positive control gene for hepatitis A virus. delete A recombinant vector comprising the hepatitis A virus standard positive control gene of claim 1. 8. The method of claim 7,
Wherein said recombinant vector has a cleavage map of Fig.
[Figure 3]

A hepatitis A virus standard positive control RNA transcript transcribed from the hepatitis A virus standard positive control gene of claim 1. 10. The method of claim 9,
The hepatitis A virus standard positive control RNA transcript is transcribed in vitro using a T7 RNA polymerase from a recombinant vector having the cleavage map of FIG. 3. Dead body.
[Figure 3]

A kit for detecting hepatitis A virus comprising the hepatitis A virus standard positive control RNA transcript of claim 9.

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