KR20130122227A - Oligonucleotide kit for detecting epstein-bar virus and ebv detecting methods using the same - Google Patents

Abstract

The present invention relates to a kit containing a primer and a probe for detecting Epstein-Bar virus (EBV) by real-time polymerase chain reaction (PCR) and a metho for detecting EBV using the same, which quickly and quantitatively detect EBV even at a very low concentration and are expected to block diseases caused by EBV.

Description

Oligonucleotide kit for detecting Epstein-Bar virus and EBV detecting methods using the same

The present invention relates to a kit for detecting Epstein-Barr virus, and to a method for identifying and quantitatively detecting the presence or absence of Epstein-Barr virus in a biological sample using the kit.

The Epstein-Bar virus (EBV), first discovered in Burkitt's lymphoma patients, is an infectious B lymphotropic virus belonging to the human herpes viridae. It causes mononucleosis and causes various clinical symptoms such as Buckyrim lymphoma, nasopharyngeal cancer, Hodgkin's disease, central nervous system B-cell lymphoma, and lymphocytic hyperplasia (Jeong-Rim Jung, Hyun-Sook Kim. Korean J Lab Med, 2000 Jun; 20 (03) 320-329)

Infectious mononucleosis is a typical clinical symptom of EBV. The characteristic characteristics of infectious mononucleosis are systemic body complications such as fatigue, helplessness, fever, sore throat, and systemic lymphadenopathy. In addition, it is known that it is important to confirm the status of infection in patients with hematologic malignancy because of its high correlation with the pathogenesis of lymphocyte proliferative diseases after the transplantation of solid organs or hematopoietic stem cells (Seungwon Jung et al. Korean J Lab). Med 2010; 30: 554-8)

Analyzing the origin of B lymphocytes involved in the development of EBV-related lymphocyte proliferative diseases is useful for analyzing the causes of infection after transplantation and clinical symptoms of transplantation.The quantitative distribution of donor EBV is associated with It may also be an important data in analyzing the relationship between the propagation potential to transplant patients (Seungwon Jung et al. Korean J Lab Med 2010; 30: 554-8).

The incidence of infection varies according to economic conditions, hygiene and geographic location. The epidemiologic relationship of infectious mononucleosis is known to be related to the age of EBV infected patients. The age of EBV infections varies depending on the hygiene environment, but the antibody positive rate is about 80% at 3 years of age, 90% before and after 20 years of age, and almost 100% in the late 20s. In many cases, early infections in adolescents cause some infectious mononucleosis (IM), and sometimes chronic EBV infection or EBV, once potentially latent, occurs during the reactivation of opportunistic B cell lymphoma (Burkey lymphoma). (Burkitt's lymphoma). During the first infection period, 10% of peripheral B lymphocytes are infected, and as a result of the host's translational response, infected lymphocytes per million lymphocytes fall to several levels and continue to life in the latent EBV genome.

Diagnosis of EBV infection is performed by a serum test using peripheral blood accompanied with clinical symptoms, and has recently been introduced into molecular biological diagnostic methods. Korean Patent No. 10-0378942 discloses a diagnostic agent capable of detecting antibodies and a method of detecting EBV antibodies in a sample as a patent for EBV diagnosis, but diagnostic methods based on these immunological assays can be quickly tested. It has the advantage that it is difficult to quantitatively detect low sensitivity and degree of infection.

Republic of Korea Patent No. 10-0378942

 Jae Rim Jung and Hyun Sook Kim. Korean J Lab Med, 2000 Jun; 20 (03) 320-329.  Seungwon Jung et al. Korean J Lab Med 2010; 30: 554-8.  Biochem. Biophys. Res. Commun. 1998, 248, 200-203.

In the diagnosis of EBV infection, it is intended to overcome the low sensitivity in detecting EBV, which is a problem as described above, and to enable quantitative detection.

 The present invention provides an Epstein-Barr virus detection kit capable of quickly and quantitatively detecting very low concentrations of Epstein-Barr virus in the diagnosis of EBV infection.

 More specifically, the oligonucleotides represented by SEQ ID NO: 1 and oligonucleotides represented by SEQ ID NO: 2 and oligonucleotides represented by SEQ ID NO: 2 may be used to detect Epstein-Barr virus using real-time polymerase chain reaction. Provided is an Epstein-Bar virus detection kit comprising a probe configured, and provides a quantitative detection method for EBV infection using the same.

As used herein, an oligonucleotide refers to a molecule composed of two or more deoxyribonucleotides or ribonucleotides such as primers and probes, and primers are oligonucleotides that are naturally occurring (eg, as restriction fragments) or synthetically prepared, Nucleic acid amplification agents, such as DNA dependent or RNA dependent polymerases, and nucleic acid strands, when placed under suitable conditions in the presence of nucleotides, which can act as a starting point for the synthesis of primer extension products complementary to the template or target sequence. .

Template DNA for EBV detection was selected as a high homology sequence as the target sequence. (GenBank Accession Number; HM366190) (SEQ ID NO: 4, see FIG. 1) Based on this, a gene of 81bp, the 1100th to 1250th sequence, was synthesized. The solution was cloned into pGEM-T-Easy vector (see Example 1).

In order to amplify the target sequence by real-time polymerase chain reaction, primers and probes were designed to select SEQ ID NOs: 1 to 3, which are excellent candidates for amplification efficiency. Low GC content that can cause nonspecific amplification, prevents thymidine from 3 ′ end, shows proper Tm value, and has the best amplification efficiency among those designed to prevent the primer from forming secondary structure It is selected.

Detection of the real-time polymerase chain reaction according to the present invention is made through fluorescence coloring, characterized in that it comprises a fluorescent dye (reporter dye) and a quencher dye (quencher dye), which is a dyeing reagent labeled on the sock end of the probe. Preferably, two selected from FAM, TAMRA, Cy3, Cy5, HEX, JOE, VIC, TET, Alexa Fluor647, Alexa Fluor660, Texas Red, Bodipy630 / 650, ROX, or BHQ1, and the 5 ′ end and 3 ′ of the probe The 5 'end of the probe is labeled with FAM or TAMRA, which is a reporter dye, and the 3' end is labeled with BHQ1, a quencher dye.

In addition, the kit of the present invention further comprises at least one composition selected from the reaction buffer, DNA polymerase, dNTP, heat-resistant pyrophosphatase, PPi, and stabilizers used in real time polymerase chain reaction in addition to the primers and probes. desirable. The composition to be used in the kit of the present invention described above can be used both as a reagent that is commercially available for medicinal or research use or directly prepared. The composition included in the kit can be all room temperature, high temperature or lyophilization. In addition, the form of the kit can be produced by loading the well-plate form or the tube in the market. The well-plate may be manufactured without limitation, such as 96 wells, 384 wells. The storage temperature of the kit can be stored at room temperature, refrigerated or frozen (below -20 ℃ or -70 ℃), but preferably when stored at -20 ℃ shelf life is one year, may vary depending on the storage conditions .

Features of the kit of the present invention can be carried out by the real-time polymerase chain reaction method, the use of the kit is characterized by having a rapid, convenient and high sensitivity of the results obtained in the EBV detection.

Epstein-Barr virus detection method according to the invention comprises the steps of (a) extracting and quantifying DNA from the sample; (b) amplifying the DNA by real-time polymerase chain reaction using the DNA obtained in step (a) and the Epstein-Barr virus detection kit according to the present invention; And (c) analyzing the expression level of the amplified viral DNA and determining the degree of EBV infection.

Extracting DNA from the sample in step (a) can be used without limitation the conventional method, can be quantified by UV measurement or can be used without limitation by conventional known methods.

Samples that can be used in the EBV detection method of the present invention include whole blood, plasma, serum, red blood cells, white blood cells, platelets, feces, urine, tears, saliva, skin, respiratory tract, intestine, external secretions of the digestive tract, spinal fluid, It is preferable to use any one selected from lymph fluid, body fluid, tissue, and the like. Most preferably using plasma or serum.

The present invention relates to a kit for detecting EBV, and to a method for detecting EBV using the kit. More specifically, the present invention provides a kit comprising a novel primer and a probe having EBV specificity in an EBV detection kit. In the method of detecting EBV using the kit according to the present invention, the degree of infection as well as whether EBV is infected or not Can be presented quantitatively.

EBV detection kit of the present invention is characterized by being manufactured by drying the reactants used in the real-time polymerase chain reaction. Compared to the case where the reactants are stored in solution, the dry reactants have a longer shelf life, can simplify the experiment process, maximize user convenience, and significantly reduce the possibility of error by the experimenter. There is an advantage. First of all, EBV detection using the EBV detection kit according to the present invention by solving the low sensitivity in the conventional EBV diagnosis can obtain quantitative results with high sensitivity, thus providing more accurate results in diagnosis of EBV infection. Enable to provide

1 shows homology of template DNA. The red area shows the location of the selected primers and probes.
2 is a view showing the results of real-time polymerase chain reaction according to the concentration of the template DNA (10 ¹ to 10 ¹⁰ (copies / rxn)) to confirm the detection range of the present invention. (a) is a real-time polymerase chain reaction graph, and (b) shows the change in Ct value according to the concentration change.
Figure 3 shows a graph of real-time polymerase chain reaction for three batches and the standard deviation (STDEV) and coefficient of variation (CV) of the obtained Ct values for the three batches in the reproducibility evaluation for the kit of the present invention.
4 is a graph showing the detection limits of the kit for detecting EBV of the present invention.
Figure 5 is a graph comparing the results of real-time polymerase chain reaction for the example of the primer and probe of the kit for detecting EBV of the present invention and the comparative group not selected candidate group.

Hereinafter, the present invention will be described in more detail with reference to examples and comparative examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited by these embodiments.

Example 1 Preparation of EBV Virus Template DNA

Template DNA for testing quantitative detection of EBV was prepared by real time PCR using the Epstein-Barr virus detection kit according to the present invention.

Template DNA for the detection of EBV was selected with a highly homologous sequence (see Fig. 1), through the homology analysis for this, select a region with no mutation as the target sequence, and standard base sequence information using a combination of the selected primers Based on (Ginbank Accession Number; HM366190), a gene of 81 bp, which is the 1100th to 1250th sequences, was synthesized (Biochem. Biophys. Res. Commun. 1998, 248, 200-203), which was used as a pGEM-T-Easy vector ( Cat: A1360, Promega, USA).

Cloning was carried out according to the experimental method provided by Promega, E. coli pluripotent cells were used HIT-DH5a (Real Biotech Corporation, Taiwan) products, and prepared vector (Real Biotech) Transformation was performed according to the experimental method provided by Real Biotech Corporation.

The prepared pluripotent cells were plated on LB plate medium coated with ampicillin, IPTG, and X-Gal, and then cultured at 37 ° C. for 16 hours. Take the white colonies on the plate after completion of the culture, incubate for 16 hours in LB liquid medium, centrifuge and discard the supernatant, and pellet the pellet using an Accuprep plasmid DNA prep kit (Bionia, Korea). Plasmid DNA was extracted from the. Plasmid DNA was measured for concentration and purity by UV spectrometer (Shimazu, Japan). It was confirmed that the purity is between 1.8 and 2.0, and the DNA copy number was calculated by the following formula based on the concentration measurement result.

DNA copy number = {6.02 × 10 ²³ × concentration} / {(3015bp + 80) × 660} [Equation 1]

(The concentration is the concentration measured in UV spectrometer (g / ml); 3015 bp is the size of the T-easy vector; 80 bp is the size of the EBV template DNA; 1bp = 660 Da.)

The template DNA copy number was calculated and then diluted 10-fold with 1 × TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) and stored at −70 ° C. until use.

Example 2. Selection of primers and probes for EBV detection

The EBV specific primers and probes were selected from the gene sequences selected in Example 1, using the highest homology, the highest amplification efficiency, and the sequence in which the amplification products were selected closest to around 100 bp. The forward primer (SEQ ID NO: 1) adopts a base range of 1145 bp to 1168 bp of standard base sequence information (GeneBank accession number; HM366190), and the reverse primer (SEQ ID NO: 2) has a base of 1207 bp to 1235 bp Sequence ranges were adopted. Adopted criteria is that the Tm value ranges from 55 ° C. to 60 ° C., and the length includes a base sequence range comprised from 80 bp to 85 bp.

Probe selection was based on the nucleotide sequence range of 1185 bp to 1206 bp of standard base sequence information (GeneBank accession number; HM366190), which can be minimized based on the homology results of FIG. It was included in the 55 ℃ to 60 ℃ range, based on whether the length is in the range of 80 bp to 150 bp. (See Table 1)

Table 1. Oligonucleotide sequences adopted as primers and probes for detecting EBV infection

Example 3. Preparation of kit for detecting EBV.

The kit for EBV detection consists of 10 × TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA), 2.5 U Taq DNA polymerase, 20 mM dNTP, thermally resistant pyrophosphatase, PPi and stable The mixture was prepared by mixing a composition consisting of an agent (α-MG, trehalose, Tween 20) and adding a primer (SEQ ID NOs: 1 and 2) and a probe (SEQ ID NO: 3) employed in Example 2. The mixture was loaded into a 96 well plate and dried to complete the kit for EBV detection.

Example 4. Evaluation of kit detection range for EBV detection

In order to evaluate the EBV detection range using the EBV detection kit prepared in Example 3, the concentration of EBV template DNA prepared in Example 1 was administered to the kit, and the total dose was 50 μl. Based on the total dose of 50 μl mixture, the concentrations of the forward primer, the reverse primer, and the probe included in the kit were 15 pmole, respectively. The detection range of the kit thus prepared was confirmed by real-time polymerase chain reaction method. Specifically, the EBV template DNA according to Example 1 was prepared in 10 sections by diluting step by step from high concentration to low concentration (1 × 10 ¹ to 1 × 10 ¹⁰ copies / rxn). The real time polymerase chain reaction was performed using an ExiCycler ^™ Real-Time PCR System (Bioner, Korea). Specifically, real-time polymerase chain reaction conditions were denatured at 95 ° C. for 5 minutes, followed by 45 cycles of 5 seconds at 95 ° C. and 5 seconds at 55 ° C., respectively. The amplified fluorescence value was continuously measured once after 55 ° C. 30 seconds reaction as each cycle proceeded. At this time, the internal control gene (tobacco mosaic virus (TMV) DNA sequence was used as the internal control gene.

Example 5 Evaluation of Reproducibility of Kits for EBV Detection

Reproducibility evaluation was repeated three times in the same manner as in Example 3. Repeated test results are shown in Table 2 and FIG. 3. As shown, the standard deviation (STDEV) and coefficient of variation (CV) were found to be valid values.

Table 2. To evaluate the reproducibility of the EBV detection kit, it shows the Ct value obtained by repeating the experiment three times for each concentration.

Example 6 Accuracy Evaluation of EBV Detection Kit

In order to evaluate the precision of the EBV detection kit according to the present invention, three levels of template DNA concentrations were selected as 10 ³ , 10 ⁵ , and 10 ⁷ copies / reaction, followed by intra-assay and inter-test. Accuracy was evaluated by calculating standard deviation (STDEV) and coefficient of variation (CV) of Ct through assay, inter-batch and 8-day repeated tests (see Table 3).

Intra-assay analysis was performed three times, three times when the same experimenter was tested once using the kit produced in the same batch (inter-assay), the inter-assay was the test Three different experimenters were analyzed by the same method as my analysis, and the inter-batch was analyzed by the same experimenter using different production batches of the kit.

Table 3. Standards of Ct obtained through intra-assay, inter-assay, inter-batch, and 8-day repeated tests to assess the precision of EBV detection kits The table shows deviation (STDEV) and coefficient of variation (CV,%).

Example 7 Evaluation of Detection Limit of EBV Detection Kit

In order to evaluate the 'detection limit' of the kit for detecting the EBV of the present invention, a panel was prepared by diluting step from high concentration to low concentration using a panel of 'The 1st WHO international standard for Epstein-Barr virus (EBV)'. In the same manner as in Example 3, an amplification component was prepared, and a panel of each concentration was injected to measure fluorescence values, and 32 repeat tests were performed for each concentration for statistical analysis. Statistical results were performed through Probit analysis, and the detectable concentration was determined as the detection limit. (See Fig. 4)

Example 8 Evaluation of Storage Stability of Kits for EBV Detection

In order to evaluate the storage stability of the kit for detecting EBV provided in the present invention, the template DNA concentration was selected as 10 ³ , 10 ⁵ , 10 ⁷ copies / reaction in the same manner as in Example 5 and stored at 40 ° C. for 7 days. Then, the performance was evaluated by real-time polymerase chain reaction in the same manner as in Example 3 at 1 day intervals. The results obtained in this experiment confirmed the predicted storage stability based on the Arrhenius equation for accelerated stability for storage temperature conditions of the product. The kit of the present invention has a shelf life of 1 year when stored below -20 ° C.

Comparative Example 1. Real-time polymerase chain reaction using a comparative group primer and probe not selected as a primer and probe for detecting EBV according to the present invention

Real-time polymerase chain reaction using the primers and probes used in the comparative example was performed in the same configuration and method as in Example 4 to compare the results.

The primer and probe sequences used in the comparative examples are as follows. In this comparison, a sequence designed to satisfy the conditions as a primer and a probe was used as a comparative example. Comparing the primers and probes adopted in the present invention, the Ct value and the intensity of the curves of the comparative example were confirmed to be inferior. (See Figure 5)

Table 4. Oligonucleotide Sequences Used as Comparative Examples.

<110> Bioneer <120> olignucleotide kit for detecting Epstein-bar virus and EBV          deteting methods using the same <130> 12P0430 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> DNA primer <400> 1 gcgagcaatc ggacatttg 19 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> DNA primer <400> 2 ggtagccgtt cgtgtgataa tga 23 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DNA probe <400> 3 ttggcacggc ccccaagaca 20

Claims (7)

An Epstein-Barr virus detection kit comprising a primer set consisting of an oligonucleotide of SEQ ID NO: 1 and an oligonucleotide of SEQ ID NO: 2 and a probe consisting of an oligonucleotide of SEQ ID NO: 3. The method of claim 1,
The kit is Epstein-Bar virus detection kit further comprises one or more selected from the reaction buffer, DNA polymerase, dNTP, heat-resistant pyrophosphatase, PPi and stabilizers used in the real-time polymerase chain reaction method. The method of claim 1,
Epstein-Barr virus detection kit, characterized in that both ends of the probe is labeled with a staining reagent. The method of claim 3, wherein
The dyeing reagent is selected from FAM, TAMRA, Cy3, Cy5, HEX, JOE, VIC, TET, Alexa Fluor647, Alexa Fluor660, Texas Red, Bodipy630 / 650, ROX or BHQ1, each of which is the 5 ′ end of the probe and Epstein-Barr virus detection kit, characterized in that labeled at the 3 'end. 5. The method of claim 4,
Epstein-Barr virus detection kit, characterized in that the 5 'end of the probe is labeled with a fluorescent reagent (reporter dye) FAM or TAMRA, and the 3' end is labeled with a BHQ1, a quencher dye (quencher dye). (a) extracting and quantifying DNA from the sample;
(b) amplifying the DNA by real-time polymerase chain reaction using the DNA obtained in step (a) and the kit for detecting Epstein-Barr virus according to any one of claims 1 to 5; And
(c) analyzing the expression level of the amplified viral DNA to determine the degree of EBV infection. The method according to claim 6,
The sample is a method for detecting Epstein-Barr virus, characterized in that the plasma or serum.

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