KR101768955B1 - Primer set for diagnosing Ebola virus and uses thereof - Google Patents
Primer set for diagnosing Ebola virus and uses thereof Download PDFInfo
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- KR101768955B1 KR101768955B1 KR1020150114451A KR20150114451A KR101768955B1 KR 101768955 B1 KR101768955 B1 KR 101768955B1 KR 1020150114451 A KR1020150114451 A KR 1020150114451A KR 20150114451 A KR20150114451 A KR 20150114451A KR 101768955 B1 KR101768955 B1 KR 101768955B1
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Abstract
The present invention relates to an Ebola virus diagnosis kit comprising an Ebola virus diagnostic primer set, an Ebola virus diagnostic kit including the primer set and a reagent for performing an amplification reaction, the Ebola virus diagnostic primer set, and a probe comprising a nucleotide sequence of SEQ ID NO: And a method for diagnosing Ebola virus using the primer set.
Description
The present invention relates to a primer set for Ebola virus diagnosis, and more particularly to a primer set for Ebola virus diagnosis, an Ebola virus diagnosis kit including the primer set and a reagent for performing amplification reaction, the Ebola virus diagnostic primer set And a probe comprising the nucleotide sequence of SEQ ID NO: 3, and a method for diagnosing Ebola virus using the primer set.
Ebola haemorrhagic fever is an infection caused by Filoviridae Ebola virus. Its mortality rate is as high as 50-90%, its natural host is unknown, and there is no vaccine or remedy. From 1976 to 2012, there were epidemics in Gabon, the Democratic Republic of Congo, Congo, Uganda, Sudan and Cote d'Ivoire in central Africa. However, as of September, 2014, there was an outbreak of Ebola in Guinea, Liberia and Nigeria And more than 1,900 deaths were reported. Ebola virus infection is generally caused by contact with the infected animal in the first patient, and secondary infection from person to person is caused by direct contact with blood or body fluids, medical treatment, and the like.
Infection with Ebola virus causes a sudden onset of incubation after 2-21 days of incubation and causes clinical symptoms such as fever, chills, headache, loss of appetite, pain in joints and muscles, and neck pain. These symptoms soon lead to nausea, vomiting, sore throat, abdominal pain and diarrhea. Patients at the onset of the disease usually have general symptoms and dehydration symptoms, and are helpless and unattractive. Usually, there are protrusions of pharyngeal and conjunctiva, and in the days to a few days there is characteristic speckle rash, petechial hemorrhage, and mucosal hemorrhage all over the body. Clinical studies show that leukopenia is rapidly progressing with secondary bacteremia, and the platelet count drops to 50,000 to 100,000 / mm 3 in the bleeding period. Early symptoms such as hemorrhage or skin rash appearing early in infection are common in patients with other diseases, and it is difficult to distinguish early diagnosis of Ebola hemorrhagic fever. Final identification of hemorrhagic fever virus is only possible with institutions with BSL4 facilities.
Korean Patent No. 1540717 discloses a one-step real-time reverse transcription polymerase chain reaction method using a probe and a primer set for detection of Ebola and Marburg virus. However, as in the present invention, a primer set for diagnosis of Ebola virus Its use has not been disclosed.
SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a rapid and sensitive biological terrorism which can diagnose Ebola virus causing Ebola hemorrhagic fever using real- And to develop a high-risk pathogen detection kit. As a result, based on the Ebola virus-specific gene sequence, the detection site was selected through homology analysis, and three primer sets were prepared based on this region. The three primer sets thus prepared were used for the detection of Ebola virus pathogen In order to diagnose the infection, real-time PCR was performed, and the present invention was completed by developing one primer set which can diagnose Ebola virus more accurately and more rapidly than any other primers.
In order to solve the above problems, the present invention provides a set of primers for diagnosing Ebola virus comprising oligonucleotide primer sets of SEQ ID NOS: 1 and 2.
Further, the present invention provides a primer set comprising: the primer set; And a reagent for carrying out an amplification reaction.
The present invention also provides a composition for real-time RT-PCR for the diagnosis of Ebola virus comprising a set of oligonucleotide primers of SEQ ID NOS: 1 and 2 and a probe comprising the nucleotide sequence of SEQ ID NO: 3.
In addition,
Isolating RNA from suspected samples of Ebola virus infection;
Performing real-time RT-PCR using the separated RNA as a template and using the primer set and the oligonucleotide probe for detecting ebola virus comprising the nucleotide sequence of SEQ ID NO: 3 to amplify the target sequence; And
And detecting the amplification product. The present invention also provides a method for diagnosing Ebola virus.
Through the present invention, a diagnostic primer set for high-risk pathogens of Ebola virus, a pathogen causing Ebola hemorrhagic fever, was selected. Through this, it is expected that it will be possible to quickly and accurately apply the diagnosis system of high-risk pathogens in the country and prepare and respond to the biological warfare (terror).
FIG. 1 shows the results of confirming the reactivity of three primer sets for the detection of Ebola virus developed by the present invention using SYBR Green reagent.
FIG. 2 is a result of a real-time PCR test using three primer sets for detection of Ebola virus developed in the present invention in order to select an optimal primer set.
FIG. 3 shows the result of measuring the minimum detection limit concentration of Ebola virus using a primer set selected in the present invention.
FIG. 4 shows the result of performing a specificity test of Ebola virus using a primer set selected in the present invention. B. anthracis : anthrax, F. tularensis : fungi, smallpox: two bacilli, Y. pestis : bacterium, Brucella: brucella
FIG. 5 shows the final test results of application of the field diagnostic equipment using the primer set developed in the present invention.
In order to achieve the object of the present invention, the present invention provides a set of primers for diagnosing Ebola virus comprising oligonucleotide primer sets of SEQ ID NOS: 1 and 2.
The oligonucleotide may preferably be an oligonucleotide consisting of fragments of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 consecutive nucleotides in the sequence of SEQ ID NO: 1 have. Also, the oligonucleotide may preferably be an oligonucleotide consisting of fragments of 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, or 21 or more consecutive nucleotides in the sequence of SEQ ID NO: 2.
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 will depend on the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.
In the present invention, the oligonucleotide used as a primer may also include a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or And may include an intercalating agent.
Further, the present invention provides a primer set comprising: the primer set; And a reagent for carrying out an amplification reaction. In the kit of the present invention, the reagent for carrying out the amplification reaction may include reverse transcriptase, DNA polymerase, dNTPs, buffer and the like.
The Ebola virus diagnostic kit according to one embodiment of the present invention may further comprise an oligonucleotide probe for detecting ebola virus, which comprises the nucleotide sequence of SEQ ID NO: 3.
As used herein, "probe" refers to a single-stranded nucleic acid sequence that hybridizes with a complementary single-stranded target sequence to form a double-stranded molecule (hybrid).
As used herein, an oligonucleotide used as a probe may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or alternatively, And may include an intercalating agent.
The present invention also provides a composition for real-time RT-PCR for the diagnosis of Ebola virus comprising a set of oligonucleotide primers of SEQ ID NOS: 1 and 2 and a probe comprising the nucleotide sequence of SEQ ID NO: 3.
In addition,
Isolating RNA from suspected samples of Ebola virus infection;
Performing real-time RT-PCR using the separated RNA as a template and using the primer set and the oligonucleotide probe for detecting ebola virus comprising the nucleotide sequence of SEQ ID NO: 3 to amplify the target sequence; And
And detecting the amplification product. The present invention also provides a method for diagnosing Ebola virus.
The suspected samples of Ebola virus infection include biological samples such as blood, serum, plasma, peripheral blood mononuclear cells, peripheral blood leukocytes, saliva, urine, feces, throat swabs, dermal lesion swabs, Cerebrospinal fluids, cervical smears, nasal juices, food matrices, and tissues of various parts of the body including the brain, spleen and liver.
The method of the present invention comprises isolating RNA from suspected samples of Ebola virus infection. As a method for separating RNA from the sample, a method known in the art can be used, and phenol: chloroform extraction followed by ethanol precipitation can be used. Using the separated RNA as a template, real-time RT-PCR was performed using an oligonucleotide primer set according to an embodiment of the present invention and an oligonucleotide probe for detecting ebola virus consisting of the nucleotide sequence shown in SEQ ID NO: 3 to obtain a target sequence Can be amplified.
The method of the present invention comprises detecting said amplification product. The detection of the amplification product can be performed through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
1. For Ebola virus screening indicating pathogenicity primer making
1) Detection site selection and lift design ( oligo design )
Based on the gene sequences in Table 1 of the Ebola virus Zaire strain, detection sites were selected through homology analysis. Gene sequencing was performed on a genbank based on reference sequence data. For the analysis of homology, MEGA (MOLECULAR EVOLUTIONARY GENETICS ANALYSIS, http://www.megasoftware.net/ ) 5.1 program was used. Ebola virus template DNA was prepared by DNA fragmentation and then transcribed with RNA using an in vitro transcription kit .
The selected primers and probe sequences were identified by sequence specificity using Basic Local Alignment Search Tool (BALST) (http://blast.ncbi.nlm.nih.gov/Blast ). Tm analysis of selected primers and probes was performed by IDT The oligonucleotide was analyzed using OligoAnalyzer 3.1 web-based program ( http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/ ) with oligonucleotide concentration of 0.25 uM and Na + concentration of 50 mM. Respectively.
size(
bp
)
2) Real - time PCR optimization
The template RNA of Ebola virus, which is a high-risk pathogen, is difficult to sample for the present invention. The template RNA of the Ebola virus is synthesized by DNA fragmentation from the homology analysis, amplified by PCR reaction, purified, Were synthesized and used. For the detection of Ebola virus, the primer reactivity was checked by SYBR Green test for each primer set, and the optimal set was selected by conducting condition test such as the limit of detection using a probe and PCR efficiency. After performing Master mixture condition test (enzyme test, buffer test) and PCR condition test using selected primer set, LOD test, dynamic range test and specificity test were performed under optimal conditions.
The SYBR Green test of the candidate primer set of Ebola virus was performed to confirm the reactivity of the primer set. The primer / probe concentration, the probe die, the PCR master mixture condition test, the PCR temperature and the reaction condition test, Respectively. Genes that determine the virulence of Ebola virus were selected and subjected to condition tests after different fluorescent labels of each gene probe for real-time PCR. The experiments were performed using Roche's LC480 Real-time PCR instrument. Table 3 shows the PCR protocol performed.
Example 1. For Ebola virus screening indicating pathogenicity primer selection( SYBR Green test )
SYBR Green reagent was used to confirm the reactivity of three types of primer sets of Ebola virus (Fig. 1). Each experiment was repeated three or more times.
Example 2. Optimal primer Set screening test
After confirming the reactivity and amplification efficiency of the primer set through the SYBR Green test, Real-time PCR test was performed to confirm the reactivity of three primer sets of Ebola virus, and then, an optimal primer set was selected. Ebola virus template RNA was diluted to a concentration of 10 7 to 10 1 copies / rxn, and the experimental results were confirmed according to the PCR efficiency, reactivity and probe concentration of each primer set.
As a result of the experiment, since the results of the Ct value, the minimum detection limit concentration, the PCR efficiency and the reactivity by concentration were the best when using the primer set 1 (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3), the primer set 1 was selected as the optimum primer set (Fig. 2). Each experiment was repeated three or more times.
Example 3. Detection minimum threshold concentration ( LOD ) Measure
The template RNA was serially diluted at a concentration of 10 7 to 10 1 copies / rxn and then the detection limit limit of detection (LOD) was measured using a selected primer set. Each experiment was repeated three or more times. As a result of measuring the minimum detection limit concentration of Ebola virus using the finally selected primer set, it was confirmed that it was effectively detected up to 10 copies / rxn (FIG. 3).
Example 4. Ebola virus specificity ( Specificity ) Test
Real-time PCR of the final set of Ebola viruses using 5 dead pathogens of other pathogens revealed that Ebola virus was not detected in all 5 species, so that the selected primer set only correctly detected Ebola virus Respectively. Each experiment was repeated three or more times. Specifically, the present invention provides a method for specifically detecting Ebola virus only in an anthrax (genomic DNA), yato (genomic DNA), pocke (DNA fragment), pest (DNA fragment), and brucella (Fig. 4).
Example 5. In the present invention, primer The final field diagnostic equipment application test using set
In order to confirm the infection in a short time in the future, many molecular diagnostic fields are being developed for the field diagnosis. In the present invention, the method of field diagnosis was studied by applying to the UltraFast LabChip Real-time PCR instrument of NanoBio CIS. The instrument is an ultra-small device with a weight of about 5.5kg. It can test 30 cycles of real-time PCR within about 15 minutes and is a chip-based PCR device that is different from conventional real-time PCR.
Ebola virus template RNA was diluted by concentration and then tested using an UltraFast LabChip real-time PCR instrument. As a result, it was confirmed that up to 10 4 copies / rxn was detected within 15 minutes of reaction time (FIG. 5). Each experiment was repeated three or more times.
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Claims (5)
The isolated RNA is used as a template and has an oligonucleotide primer set of SEQ ID NOS: 1 and 2 and has a detection limit of detection (LOD) of 10 copies / reaction and has an anthrax, Real-time RT-PCR was performed using a primer set for on-site detection of Ebola virus and an oligonucleotide probe for Ebola virus detection consisting of the nucleotide sequence of SEQ ID NO: 3, characterized in that only the Ebola virus was specifically detected without detecting the brucella target Amplifying the target sequence; And
And detecting the amplification product.
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