KR101678553B1 - Oligonucleotide kit for detecting Epstein-Bar virus and EBV detecting methods using the same - Google Patents

Abstract

The present invention relates to an EBV detection kit comprising primers and probes that can be used to detect Epstein-Bar virus (EBV) using real-time polymerase chain reaction (PCR) The present invention relates to a detection method using the same, and it is possible to quantitatively detect Epstein-Barr virus to a very low concentration in a quantitative manner, and it is expected to have an effect of blocking EBV disease.

Description

A kit for detecting Epstein-Barr virus and a method for detecting Epstein-Bar virus using the same,

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

Epstein-Bar virus (EBV), first discovered in patients with Burkitt's lymphoma, is a B lymphotropic virus belonging to the human herpesvirus, It has been reported that the clinical manifestations of various clinical symptoms such as Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, CNS lymphoma, lymphoproliferative disease are becoming increasingly important as they cause mononucleosis (Jung Jim Jung, 2000 Jun; 20 (03) 320-329)

Infectious mononucleosis is a typical clinical manifestation of EBV. Characteristic of infectious mononucleosis is systemic physical complication, resulting in fatigue, weakness, fever, sore throat, and systemic lymphadenopathy. In addition, it is known that it is important to confirm the status of infection in hematologic tumor patients because of the high correlation with pathogenesis of lymphoproliferative diseases after transplantation of solid organs or hematopoietic stem cells (Seungwon Jung et al. Med 2010; 30: 554-8)

The analysis of the origin of B lymphocytes involved in the development of EBV-associated lymphoproliferative disorders is useful for analyzing the etiology of clinical signs and symptoms after transplantation and the quantitative distribution of donor EBV is related to the EBV load (Jung et al., 2002), and the role of the transplantation in the development of transplantation.

The incidence of infection varies according to economic conditions, hygiene status and geographical location. The epidemiological relationship of infectious mononucleosis is known to be related to the age of EBV infected patients. The EBV infection rate differs depending on the hygienic environment, but the positive rate is 80% at 3 years, 90% at around 20 years, and almost 100% in late 20s. When infected with infancy, the symptoms become asymptomatic or mild However, EBV infection, which causes chronic infectious mononucleosis (IM) and sometimes chronic infectious EBV infection or infection, may cause opportunistic B cell lymphoma, Burkitt's lymphoma (Burkitt's lymphoma). At the time of primary infection, 10% of peripheral B lymphocytes are infected, and as a result of host transformation, infected lymphocytes per million lymphocytes fall to several levels and last for a lifetime as a latent EBV genome.

The diagnosis of EBV infection is made by serologic tests using peripheral blood with clinical symptoms and has recently been introduced into molecular biologic diagnosis. Korean Patent No. 10-0378942 discloses a patent for EBV diagnosis and a method for detecting an antibody capable of detecting an antibody and a method for detecting an EBV antibody in a sample. However, the diagnostic methods based on the immunological method can be quickly tested However, there is a limitation in that it is difficult to detect quantitatively the sensitivity and the degree of infection.

Korean Patent No. 10-0378942

 Purpose: 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 low sensitivity in EBV detection, which is a problem as described above, and to perform quantitative detection.

 The present invention provides a kit for detecting Epstein-Bar virus capable of rapidly quantitatively detecting Epstein-Barr virus at a very low concentration due to its high sensitivity in the diagnosis of EBV infection.

 A primer consisting of the oligonucleotide described in SEQ ID NO: 1 and the oligonucleotide described in SEQ ID NO: 2 and the oligonucleotide described in SEQ ID NO: 3, which can be used for detecting Epstein-Barr virus using real-time polymerase chain reaction Provided is a kit for detecting Epstein-Bar virus including a probe, and provides a quantitative detection method for EBV infection using the kit.

Oligonucleotides used in the present invention refer to molecules composed of two or more deoxyribonucleotides or ribonucleotides such as primers and probes, wherein the primers are oligonucleotides that are naturally occurring (e.g., as restriction sites) or synthetically produced oligonucleotides, Refers to a nucleic acid strand that can act as a starting point for the synthesis of a primer extension product complementary to a template or target sequence when placed under suitable conditions in the presence of nucleic acid amplifiers such as DNA-dependent or RNA-dependent polymerases and in the presence of nucleotides .

The template DNA for EBV detection was selected as the target sequence with high homology (Jinbank accession number: HM366190) (SEQ ID NO: 4, see Fig. 1). Based on this, 81bp gene of the 1100th to 1250th sequence was synthesized And cloned into the pGEM-T-Easy vector (see Example 1).

Primers and probes were designed to amplify the target sequence in a real-time PCR, and SEQ ID NOS. 1 to 3, which are candidates with excellent amplification efficiency, were selected. The GC content that can cause nonspecific amplification is lowered, the thymidine does not come to the 3 'end, the proper Tm value is shown, and the primer is designed so as not to form the secondary structure, .

The detection of the real-time PCR reaction according to the present invention is performed through fluorescence coloring and includes a reporter dye and a quencher dye which are labeled at both ends of the probe. Two species are selected from among FAM, TAMRA, Cy3, Cy5, HEX, JOE, VIC, TET, Alexa Fluor 647, Alexa Fluor 660, Texas Red, Bodipy 630/650, ROX or BHQ1, , More preferably the 5 'end of the probe is labeled with a reporter dye, FAM or TAMRA, and the 3' end is labeled with a quencher dye, BHQ1.

In addition, the kit of the present invention may further comprise at least one composition selected from a reaction buffer solution, DNA polymerase, dNTP, heat-resistant pyrophosphatase, PPi, and a stabilizer used in the real-time PCR in addition to the primer and the probe desirable. The composition for use in the kit of the present invention can be used either as a reagent commercially available for medicinal use or for research, or as a composition for direct use. The composition contained in the kit may be room temperature, high temperature or freeze-dried. In addition, the kit can be in the form of a commercially available well-plate or tube. The well-plate can be fabricated without limitation, such as 96 wells and 384 wells. The storage temperature of the kit can be stored at room temperature, refrigerated or frozen (-20 ° C or -70 ° C or less), preferably at -20 ° C, and the shelf life is one year. .

The kit of the present invention can be characterized by real-time PCR, and the use of the kits described above is characterized by rapidity of acquisition, convenience, and high sensitivity in EBV detection.

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

The method of extracting DNA from the sample in step (a) can be used without limitations in the conventional method, and can be quantitatively determined by UV measurement or can be used without limitation in known methods known in the art.

The sample that can be used in the EBV detection method of the present invention may be a whole blood, plasma, serum, red blood cells, white blood cells, platelets, feces, urine, tears, saliva, skin, respiratory tract, Lymph fluid, body fluid, tissue, or the like is preferably used. Most preferably plasma or serum.

The present invention relates to an EBV detection kit, and a method of detecting EBV using the kit. More particularly, the present invention provides a kit comprising a novel primer and a probe having EBV specificity in an EBV detection kit, and in the method of detecting EBV using the kit according to the present invention, not only EBV infection but also the degree of infection Can be quantitatively presented.

The EBV detection kit of the present invention is characterized in that the EBV detection kit is prepared by drying a reaction product used in real-time PCR. As compared with the case where the reactants are stored in a solution state, the kit can maximize the user convenience by simplifying the experimental procedure and the possibility of errors due to the operator errors, There are advantages. Above all, since EBV detection using the EBV detection kit according to the present invention solves the low sensitivity in the conventional EBV diagnosis, it is possible to obtain a quantitative result with a high sensitivity, so that a more accurate result is obtained in the diagnosis of EBV infection .

Fig. 1 is a diagram showing the homology of the template DNA. The area marked in red indicates the location of the selected primer and probe.
FIG. 2 is a diagram showing the results of a real-time PCR reaction according to the concentration (10 ¹ to 10 ¹⁰ (copies / rxn)) of the template DNA in order to confirm the detection range of the present invention. (a) is a real time PCR chain reaction graph, and (b) is a graph showing a change in Ct value according to concentration change.
Figure 3 shows a graph of the real time PCR reaction for three batches with the reproducibility evaluation for the kit of the present invention and the standard deviation (STDEV) and the coefficient of variation (CV) of the obtained Ct values.
4 is a graph showing detection limits of the EBV detection kit of the present invention.
FIG. 5 is a graph comparing the results of real-time PCR for the primers and probes of the EBV detection kit of the present invention and the non-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 the quantitative detection of EBV was prepared by real-time PCR using a kit for detecting Epstein-Barr virus according to the present invention.

The template DNA for EBV detection was selected with high homology (see FIG. 1). Through the homology analysis of the template DNA, the region without the mutation was selected as the target sequence, and using the combination of the selected primers, (Biochem. Biophys. Res. Commun. 1998, 248, 200-203) was synthesized using the pGEM-T-Easy vector (GenBank Accession No. HM366190) Cat: A1360, Promega, USA).

E. coli competent cells were obtained from HIT-DH5a (Real Biotech Corporation, Taiwan), and the prepared vector was transferred to Real Biotechnology Co., Ltd. Transformation was carried out according to the experimental method provided by Real Biotech Corporation.

The prepared pluripotent cells were plated on LB plate medium coated with ampicillin, IPTG, X-Gal, and cultured at 37 ° C for 16 hours. The white colony on the culture plate was taken and cultured for 16 hours in LB liquid medium. After centrifugation, the supernatant was discarded and the pellet was extracted using a plasmid DNA preparation kit (Bioneer, Korea) The plasmid DNA was extracted from the plasmid DNA. Plasmid DNA was measured for concentration and purity using a UV spectrometer (Shimazu, Japan). The purity was confirmed to be between 1.8 and 2.0, and the copy number of DNA was calculated by the following formula based on the result of the concentration measurement.

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

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

The number of template DNA copies was calculated and then diluted 10 times with 1X 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

Selection of EBV-specific primers and probes was performed using the sequences selected from the gene sequences selected in Example 1 that have the highest homology, the highest amplification efficiency, and the amplification products that can be selected closest to about 100 bp. The forward primer (SEQ ID NO: 1) adopts the base sequence range of 1145 to 1168 bp of the standard sequence information (GeneBank Accession No. HM366190) and the reverse primer (SEQ ID NO: 2) adopts the base sequence of 1207 to 1235 bp Sequence coverage was adopted. Adoption criteria include those in which the Tm value is in the range of 55 占 폚 to 60 占 폚 and the length is in the range of 80 to 85 bp.

Selection of the probes employs a base sequence range of 1185 bp to 1206 bp of the standard sequence information (GeneBank Accession No. HM366190), which can minimize the base mutation based on the homology result in Fig. 1, Was included in the range of 55 占 폚 to 60 占 폚, and the length was included in the range of 80 to 150 bp. (See Table 1)

Table 1. Primer to detect EBV infection and oligonucleotide sequence adopted as probe

Example 3 Preparation of EBV detection kit.

EBV detection kit was prepared using 10 × TE buffer solution (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA), 2.5 U Taq DNA polymerase, 20 mM dNTP, thermostable pyrophosphatase, PPi and stable (Α-MG, trehalose, Tween 20) were mixed, and the mixture was prepared by adding the primers (SEQ ID NOs: 1 and 2) and the probe (SEQ ID NO: 3) adopted in Example 2 above. The mixture was loaded on a 96-well plate and dried to complete the kit for EBV detection.

Example 4 Evaluation of detection range of kit for EBV detection

In order to evaluate the EBV detection range using the EBV detection kit prepared in Example 3, the EBV template DNA of each concentration prepared in Example 1 was administered to the kit so that the total volume was 50 μl. The concentration of the forward primer, the reverse primer and the probe contained in the kit was 15 pmole, respectively, when the total volume was 50 μl. The detection range of the prepared kit was confirmed by real-time PCR. Specifically, the EBV template DNA according to Example 1 was diluted stepwise from a high concentration to a low concentration to prepare 10 sections (1 × 10 ¹ to 1 × 10 ¹⁰ copies / rxn). Real-time PCR was performed using Exisycler real-time PCR (ExiCycler ^™ Real-Time PCR System, Bioneer, Korea). Specifically, the real-time PCR reaction conditions were denatured at 95 ° C for 5 minutes, followed by 45 cycles of reaction at 95 ° C for 5 seconds and at 55 ° C for 5 seconds. The amplified fluorescence value was continuously measured once at 55 ° C for 30 seconds as the cycle progressed. (See FIG. 2). At this time, the internal control gene used the tobacco mosaic virus (TMV) DNA sequence.

Example 5: Evaluation of reproducibility of EBV detection kit

The reproducibility evaluation was repeated three times in the same manner as in Example 3. The results of the repeated experiments are shown in Table 2 and FIG. As shown, the standard deviation (STDEV) and the coefficient of variation (CV) were valid values.

Table 2 shows the Ct values obtained by repeating the experiment three times for each concentration in order to evaluate the reproducibility of the EBV detection kit.

Example 6. Evaluation of precision of EBV detection kit

In order to evaluate the accuracy of the EBV detection kit according to the present invention, the template DNA concentration in the three stages was selected as 10 ³ , 10 ⁵ , and 10 ⁷ copies / reaction, and intra-assay, inter- (STDEV) and coefficient of variation (CV) were calculated and evaluated for accuracy (see Table 3), using the inter-assay, inter-batch and 8-

The intra-assay was repeated three times when the same experiment was performed once using the same batch of the kit, and the inter-assay was carried out in the same manner as described above In the same way as my analysis, three different experimenters were analyzed, and inter-batch analysis was performed by the same experimenter using different production batches of the kit.

Table 3. Standard of Ct obtained through intra-assay, inter-assay, inter-batch and 8-day repeated testing for the precision evaluation of EBV detection kit (STDEV) and coefficient of variation (CV,%).

Example 7. Detection limit of EBV detection kit

In order to evaluate the 'detection limit' of the EBV detection kit according to the present invention, it was prepared by diluting the EBV with a high concentration at a low concentration using a 'The 1st WHO International Standard for Epstein-Barr virus (EBV)' panel. Amplification constructs were prepared in the same manner as in Example 3, fluorescence values were measured by injecting panels of respective concentrations, and 32 repeated tests were performed for each concentration for statistical analysis. The results obtained from the experiment were analyzed by Probit analysis, and the detection limit of 95% or more was defined as the detection limit. (See Fig. 4)

Example 8. Evaluation of Storage Stability of EBV Detection Kit

In order to evaluate the storage stability of the EBV detection kit provided in the present invention, the template DNA concentration was selected as 10 ³ , 10 ⁵ , and 10 ⁷ copies / reaction in the same manner as in Example 5 and stored at a temperature of 40 ° C. for 7 days And the performance was evaluated by performing a real-time PCR in the same manner as in Example 3 at intervals of one day. The results obtained in this experiment confirmed the storage stability predicted based on the Arrhenius equation for the accelerated stability for the storage temperature condition of the product. The kit of the present invention was adopted for a period of one year when stored below -20 ° C.

Comparative Example 1. Real-time polymerase chain reaction (PCR) using primers and probes of Comparative Group not selected as primers and probes for EBV detection according to the present invention

The real-time PCR reaction using primers and probes used in the comparative examples was carried out by the same constitution and method as in Example 4, and the results were compared.

The primer and probe sequences used in the comparative examples are as follows. In this comparison, a designed sequence satisfying the conditions as a primer and a probe was used as a comparative example. Compared with the primers and probes adopted in the present invention, it was confirmed that the Ct value of the comparative example and the intensity of the curve were inferior. (See Fig. 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)

A kit for detecting Epstein-Barr virus comprising a primer set consisting of an oligonucleotide represented by SEQ ID NO: 1 and an oligonucleotide represented by SEQ ID NO: 2, and a probe comprising an oligonucleotide represented by SEQ ID NO: The method according to claim 1,
Wherein the kit further comprises at least one selected from a reaction buffer solution, DNA polymerase, dNTP, heat-resistant pyrophosphatase, PPi, and a stabilizer used in a real-time PCR. The method according to claim 1,
Characterized in that both ends of the probe are labeled with a staining reagent. The method of claim 3,
Two of the above dyeing reagents are selected from FAM, TAMRA, Cy3, Cy5, HEX, JOE, VIC, TET, Alexa Fluor 647, Alexa Fluor 660, Texas Red, Bodipy 630/650, ROX or BHQ1, 3 &apos; terminal of said Epstein-Barr virus. 5. The method of claim 4,
Wherein the 5 'end of the probe is labeled with a reporter dye, FAM or TAMRA, and the 3' end is labeled with a quencher dye, BHQ1. (a) extracting and quantifying DNA from a specimen;
(b) amplifying the DNA by real-time PCR 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 virus DNA to determine the degree of EBV infection. The method according to claim 6,
Wherein the specimen is plasma or serum.

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