KR100337194B1 - Internal Standard RNA and its Application to the Diagnosis of Enteroviral Diseases - Google Patents

Abstract

The present invention relates to an internal standard RNA, a composition for diagnosing enteric viral diseases, and a diagnostic method, and more particularly, in the process of diagnosing enteric viral genes using RT-PCR (Reverse Transcriptation-PCR). The present invention relates to an internal standard RNA corresponding to a nonstructural region of the 5 'terminal which is commonly present in the enterovirus used and a diagnostic method using the same. The method of selecting a sample for diagnosis by excluding false negatives, which was a problem of the conventional RT-PCR diagnostic method It has the effect of expanding the scope to provide the opportunity to select a variety of samples and to enable the rapid diagnosis of enteroviral diseases.

Description

Internal standard RNA and its Application to the Diagnosis of Enteroviral Diseases}

The present invention relates to an internal standard RNA, a composition for diagnosing enteric viral diseases and a diagnostic method, and more particularly, in the process of diagnosing enteric viral genes using RT-PCR (Reverse Transcriptation-PCR). The internal standard RNA used and a diagnostic method using the same.

Enterovirus is a single-stranded (+) RNA virus belonging to the family of Picornaviridae. It is a pathogen that causes aseptic meningitis, epidemic keratoconjunctivitis (Apollo eye disease), and hand and foot disease in children and infants. It is a representative human pathogen divided into serotypes.

On the other hand, the conventional methods used for the diagnosis of enteroviral diseases include isolation and identification of viruses through cell culture (Kim et al. 1994. Journal of Korea Society of Virology 24: 77-86), or RT-PCR Genetic diagnosis using methods has been used (Zoll et al. 1992. Journal of Clinical Microbiology 30: 160-165).

However, the isolation of the virus through cell culture requires a high level of cell culture technology, and there is a limitation in its usefulness because there is a Coxsackie A group virus that cannot be diagnosed by cell culture because it is not sensitive to cultured cells. In addition, since the time required for virus isolation is more than three weeks, it is not possible to provide timely data useful for treating patients.

Therefore, RT-PCR diagnostic method was developed to confirm the presence of virus by rapidly amplifying the gene of intestinal virus regardless of the cell susceptibility. On the side, there is a significant effect.

However, the series of enzymes and reaction environment used in the RT-PCR process may lead to contradictory results, ie, false negatives, which are inconsistent with clinical symptoms depending on the presence or absence of enzymatic inhibitory elements in the selected material.

Therefore, in order to avoid the error of diagnosis according to the false negative, the patient's specimen for RT-PCR is used only because the cerebrospinal fluid which is relatively uncontaminated is difficult to use the feces generally used for the diagnosis of intestinal viral diseases. There is a problem that the selectivity of the sample is very low.

The present inventors can exclude false negatives when using the standard RNA as a positive control, which includes a nucleotide sequence that binds to a primer used for the diagnosis of enterovirus disease in order to solve the problems of the conventional RT-PCR diagnostic method described above. By confirming that the present invention was completed.

Accordingly, it is an object of the present invention to provide an intrinsic standard RNA used as a positive control in RT-PCR diagnostics.

Another object of the present invention to provide a novel virus Echo30-Kor / 97 (KFCC-11096) used in the production of the internal standard RNA.

It is another object of the present invention to provide a method for diagnosing enteroviral diseases characterized by the additional addition of the internal standard RNA.

1 is a schematic diagram illustrating a cloning method of intrinsic standard RNA;

Figure 2 is a figure confirming the nucleotide sequence and the primer binding position of the cloned template DNA used for the production of internal standard RNA,

Figure 3 shows the electrophoresis of a specific product of 704bp by performing RT-PCR using primers used for the diagnosis of intestinal virus by in vitro transcription of the standard RNA, M: 1kb ladder, N: negative control, lane 1: 436 bp (E30 RNA), lane2: 704 bp (internal standard RNA),

4 is a photograph showing the 704bp specific product obtained by performing electrophoresis on RT-PCR by directly treating the internal standard RNA to the cultured positive or negatively confirmed fecal sample, M: 1kb ladder, N: negative control , lane1: negative feces, lane2: positive feces,

Figure 5 is a picture of performing RT-PCR by the concentration of RNA to analyze the detection limit of the internal standard RNA, M: 1kb ladder, N: negative control, lane 1: 500fg, lane 2: 100fg lane 3: 50fg, lane 4: 10fg, lane5: 0fg

Figure 6 is a schematic diagram showing the flow of a series of RT-PCR process and treated with the internal standard RNA to fecal specimens.

The present invention features a novel virus Echo30-Kor / 97 (KFCC-11096), which is used for the preparation of internal standard RNA added to RT-PCR diagnostics.

The present invention is characterized by an internal standard RNA having a nucleotide sequence set forth in SEQ ID NO: 6 which is used as a positive control in RT-PCR diagnostics.

The present invention further features a recombinant plasmid pE30-5F having a cleavage map shown in FIG. 1 used for cDNA of said intrinsic standard RNA and its cloning.

In the RT-PCR diagnostic method comprising extracting RNA from fecal and cerebrospinal fluid and synthesizing cDNA and PCR using the RNA as a template, RT-intestinal viral disease is characterized by additionally adding endogenous standard RNA. PCR diagnostics are another feature.

The present invention is described in detail as follows:

First, enteric viruses are isolated and identified from domestic enteroviral patients in order to produce internal standard RNA, which is particularly useful for diagnosing enteric viral diseases in Korea. Subsequently, the RNA genome of the isolated enterovirus Echo30-Kor / 97 (KFCC-11096) was isolated and purified, and its cDNA was prepared. Using primers that can selectively amplify non-structural regions at the ends, amplify DNA fragments of a certain size and clone them into TA vectors. In addition, a portion of the DNA encoding the capsid protein commonly found in enteric viruses (VP3-VP1 adjacent region) was amplified using the genome of Echo7-Kor / 95 (KCCM-10162) and cloned into a TA vector. DNA fragments are isolated by cleaving a portion with restriction enzymes.

Then, the isolated DNA fragments are linked, a recombinant plasmid containing the same is prepared, and the internal standard RNA of the present invention is prepared by in vitro transcription thereof.

The reason for selecting the non-structural portion of the 5 'end and the portion of the gene encoding the portion of the capsid protein when constructing the internal standard RNA of the present invention is that the base sequence of the non-structural part of the 5' end is relatively conserved in most enteroviruses. This is because it is used to diagnose intestinal viruses. Second, when the size of the DNA fragment of the gene portion encoding a portion of the capsid protein increases with the size of the non-structural portion of the 5 'end, the 5'-end non-structural portion differs from the amplified DNA band of 436 bp. Because it is easy to observe.

On the other hand, the diagnostic method of the enteroviral disease of the present invention is RT-PCR diagnostic method comprising the step of extracting RNA from fecal and cerebrospinal fluid and synthesizing cDNA and PCR using the RNA template, the addition of an internal standard RNA It features. In the above diagnostic method, the intrinsic standard RNA serves as a positive control, and thus, by confirming a band of the intrinsic standard RNA amplified as a result of performing the diagnostic method, it is possible to prevent an error of the result due to the conventional false negative.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example 1 Isolation and Identification of Domestic Enteric Viruses

Single-cultured RD-cdc (ATCC CCL-136) and HEp-2c (ATCC CCL-23) cells were inoculated (0.2 ml / 25 cm 2 flask) with a 10% suspension of the specimen treated with chloroform, and then cytopathic effect, CPE), and when 70-80% cell lesions were observed, the virus was isolated by freezing and thawing the cells at -20 ° C. and repeating freezing / thawing 2-3 times and then inoculating the same cells with the suspension. It was.

If a rapid CPE phenomenon is observed within 24 hours, it is considered that the toxicity of the specimen remains, and once again frozen / thawed at -20 ° C, the serum of the intestinal virus is reinoculated into RD-cdc and Hep-2c cells. All toxic effects were excluded before starting serotyping.

Serum used in the neutralization test for identification of virus was obtained from WHO using an enterovirus serum pool produced by Riksksinstituut voor volksgenzonheid (RIVM, The Netherlands). Antisera was used diluted 20 times with DMEM (Gibco BRL) containing 2% FBS. 50 μl of each antiserum (AG, polio pool, coxsack B pool) prepared in each well of a 96 well plate was added, and 50 μl of virus suspension diluted to 100-1000 TCID ₅₀ / ml was added and mixed for 1 minute. The reaction was neutralized for 1 hour and 30 minutes at ℃. Subsequently, the RD-cdc cells were 1.5 × 10 ⁵ cells / ml and 100 μl / well were dispensed and cultured, followed by observing CPE under a microscope. The serum pool of the Coxsackie B group was diluted 50-fold, and the cell culture medium neutralized in the Coxsaki serum pool was re-identified using a serum pool (Denka Seiken) subdivided into Coxsaki B1-B6.

Among the enteroviruses identified and identified by the present invention, Echo30-Kor / 97 is a depository institution of the Republic of Korea, and was deposited on May 24, 1999 to the Korean spawn management association, an international depository institution under the Budapest Treaty, and was given accession number KFCC-11096. , Echo7-Kor / 95, was deposited on May 24, 1999 by the Korean Deposit Control Association, which was the depository, and was given accession number KCCM-10162.

Example 2: Cloning of Template DNA for Preparation of Intrinsic Standard RNA

First, the RNA genome of Echo30-Kor / 97 (KFCC-11096) was isolated and purified as follows:

Rd, 500 μl of Echo30-Kor / 97 culture inoculated on HEp-2 cells were centrifuged at 14000 rpm. Subsequently, after removing the cells and their residues, RNA was extracted with 15 µl of 20% SDS, 10 µl of proteinase K (10 mg / ml), and 400 µl of phenol / chloroform / isoamyl alcohol (25/24/1). Precipitation was performed by adding 1 ml of ethanol. The precipitated RNA was then dissolved in 10 μl of diethylpyrocarbonate (DEPC) -water containing Rnasin (Promega) and stored at −70 ° C. 5 μl of the RNA sample was prepared using 5 μl of 2.5 mM dNTP, 4 μl of 5xRTase buffer solution, 2 μl of 0.1M DTT, 1 μl of MMLV (Gibco BRL), and 1 μl of random-hexamer, followed by 20 distilled water. ΜL was titrated and left at 42 ° C. for 2 hours to prepare cDNA. Then, using the prepared cDNA as a template, an omnidirectional primer capable of selectively amplifying 5 'terminal nonstructural sites commonly present in various enteroviruses (see SEQ ID NO: 1; ENTF1; manufactured by Gibco BRL) G) and reverse primer (see SEQ ID NO: 2; ENTR; produced by Gibco BRL) for 3 minutes at 94 ° C for 3 minutes and 35 times with 94 ° C for 1 minute, 52 ° C for 1 minute 30 seconds and 72 ° C for 1 minute 30 seconds. After repeating, it was finally extended for 7 minutes at 72 ° C. to amplify 436 bp DNA fragments (DNA Thermal Cycler 480, Perkin Elmer). Subsequently, the amplified 436 bp DNA fragment was cloned into a pCR2.1 vector (Invirtogen) using a TA cloning kit (Invirtogen) and the inserted DNA fragment was a DNA purification system (Wizard Plus SV Minipreps DNA purification system, Promega). Plymid was extracted with EcoRI restriction enzyme and electrophoresed on agarose gel. Thus, the synthesized vector was named pE30-2.

Meanwhile, the cDNA of Echo7-Kor / 95 was prepared in the same manner as in the above-mentioned method for preparing Echo30-Kor / 97 cDNA, and the cDNA thus prepared was used as a template, and the forward primer E7-488F (see SEQ ID NO: 3) and the reverse primer were prepared. PCR reaction solution was prepared by adding E7-488R (see SEQ ID NO: 4), dNTP 0.375 mM, MgCl ₂ 2.5 mM and Taq DNA polymerase 1.5U. Subsequently, the reaction solution was subjected to 35 cycles of total denaturation, 94 ° C. 1 minute, 55 ° C. 1 minute, and 72 ° C. 1 minute for 35 minutes at 94 ° C. using a PCR instrument (DNA Thermal Cycler 480, Perkin Elmer). Was performed. Thus, the amplified DNA was digested with EcoRV 5U and HaeIII 5U, subjected to agarose electrophoresis, and then a band of 268 bp was cut and separated using a Qiaquick Gel Extraction Kit (Qiagen).

Then, the 268 bp fragment and the pE30-2 cleaved fragment with the restriction enzyme PmaCI 5U were linked by blunt-ended ligation (Sambrook et al., Molecular Cloning a laboratory manual. 2nd Ed. 1.56-1.59), This recombinant plasmid was named pE30-5F. Subsequently, the sequence was analyzed by an automatic sequencing method using an ABI PRISM Die Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) to identify the inserted DNA fragment (see SEQ ID NO: 5).

Then, transformed XL1-blue (Takara) with pE30-5F and named the produced transformant as pE30-5F / XL1-blue, which was a Korean depository institution and an international depository institution under the Budapest Treaty. Was deposited on 24 May 1999 and was assigned accession number KFCC-11093.

Example 3: Identification of RNA Transcripted and Prepared In Vitro

The pE30-5F / XL1-blue (KFCC-11093) was incubated at 37 ° C. for 16 hours in an LB medium containing ampicillin at a concentration of 50 μg / ml, followed by a DNA purification system (Wizard Plus SV Minipreps DNA purification system, Promega) PE30-5F was isolated and purified.

The purified pE30-5F was digested with restriction enzyme BamHI 5U and purified by phenol / chloroform extraction and ethanol precipitation. Subsequently, the purified DNA was used as a template and in vitro transcription was performed using an RNA production system (RiboMax large scale RNA production system, Promega).

The prepared RNA was confirmed purity and size through formaldehyde / agarose electrophoresis, the amount was calculated by measuring the absorbance at 260nm with a spectrophotometer.

The prepared RNA was added to 100 µl of 10% PBS-fecal suspension, and 300 µl of Tri-reagent LS (Molecular Research Center) was added thereto, and the mixture was allowed to stand at room temperature for 15 minutes, followed by 80 µl of chloroform. And thoroughly mixed and left for 10 minutes at room temperature.

Subsequently, after centrifugation at 4 ° C. and 14000 rpm for 15 minutes, the colorless aqueous layer was separated and transferred to a new tube. After adding 200 μl of isopropanol, the mixture was left at room temperature for 10 minutes and centrifuged at 4 ° C. and 14000 rpm for 15 minutes. Washed with% ethanol and dried. The precipitate was then suspended in distilled water treated with 10 [mu] l DEPC for cDNA synthesis.

Then, an omnidirectional primer capable of selectively amplifying the 5 'terminal nonstructural site commonly present in various enteroviruses using a template of 1/20 of the prepared cDNA (see SEQ ID NO: 1; ENTF1; manufactured by Gibco) BRL) and reverse primers (see SEQ ID NO: 2; ENTR; manufactured by Gibco BRL) at 94 ° C for 3 minutes and denature 94 ° C for 1 minute, 52 ° C for 1 minute 30 seconds and 72 ° C for 1 minute 30 seconds. After repeating for a long time and finally extended to 72 ℃ 7 minutes to amplify the DNA fragment of 436bp (DNA Thermal Cycler 480, Perkin Elmer).

As a result of the PCR, the RNA was amplified by cDNA, and the results confirmed that the RNA prepared in the present invention could act as an enterovirus-specific internal standard RNA.

Example 4 Identifying Detection Limits of Intrinsic Standard RNA and Viral RNA Applied to RT-PCR Diagnostics

To the 10% fecal suspension diluted with PBS, the standard RNA prepared in Example 3 was added at various concentrations (21 fM-0.42 fM, 6,000-120 RNA molecules) and the same method as in the RT-PCR diagnostic procedure. The amount of RNA to be added to the actual fecal suspension was determined.

In addition, intestinal virus (Echo30-Kor / 97) diluted in various concentrations was added to the 10% stool suspension and the detection limit of the viral RNA was confirmed by the same method as the RT-PCR procedure of the internal standard RNA.

As a result, 704bp of bands were observed up to 10fg of RNA, RNA extraction and RT-PCR, and 436 bp up to 10 ³ TICD ₅₀ / mL were observed for virus.

Therefore, it was found that the limit of detection of the intrinsic standard RNA of the present invention was at least 10 fg (0.42 fM) or less, and the actual concentration of the intrinsic standard RNA was stained with ethidium bromide after agarose gel electrophoresis in actual RT-PCR diagnosis. When determined as 100fg (4.2fM) that can accurately represent the DNA band.

Example 5: Confirmation of the method via blind test

Stool confirmed the presence of virus by cell culture (Kim et al. 1994. Journal of Korea Society of Virology 24: 77-86) to confirm the reliability of the RT-PCR diagnostic method using the internal standard RNA prepared in Example 3 RT-PCR was carried out in the same manner as in Example 4 by adding 50 fg of internal standard RNA to the suspension, and as a result, the same result as that obtained by the cell culture method was obtained.

Therefore, the reliability of the RT-PCR diagnostic method using the internal standard RNA of the present invention was confirmed.

As described in detail above, the present invention provides a standard RNA for use in the RT-PCR diagnosis of enteroviral diseases. On the other hand, the present invention provides a method for diagnosing enteric viral diseases, characterized in that the addition of the endogenous standard RNA. Invention standard RNA of the present invention can exclude false negatives, which has been a problem of the conventional RT-PCR diagnostic method, thereby providing an opportunity to select various specimens by expanding the range of sample selection used for diagnosis, thereby enabling rapid diagnosis of intestinal viral diseases. It's effective.

Claims (8)

Echo30-Kor / 97 (KFCC-11096) causing enteroviral disease. DNA having a nucleotide sequence set forth in SEQ ID NO: 5. Recombinant plasmid pE30-5F comprising the nucleotide sequence set forth in SEQ ID NO: 5 and having a cleavage map shown in FIG. New strain pE30-5F / XL1-blue (KFCC-11093) transfected with the recombinant plasmid pE30-5F of claim 3. RNA having a nucleotide sequence set forth in SEQ ID NO: 6. (delete) (delete) (delete)