KR20200026356A - Primers and probes for differentiation between Rabies virus and vaccine strains and differentiating method between Rabies virus and vaccine strains using the same - Google Patents

Abstract

The present invention relates to a primer and a probe for discriminating rabies virus (RABV) and vaccinia rabies glycoprotein (V-RG), genetically modified rabies vaccine (ERAGS), or recombinant adenoviruses expressing the glycoprotein of rabies (Ad-0910G); and a method for discriminating RABV and V-RG, ERAGS, or Ad-0910G using the same. More specifically, according to the present invention, a primer and probe set can simultaneously and specifically discriminate and detect RABV and V-RG, ERAGS, or Ad-0910G and provide excellent sensitivity, thereby being useful in discrimination of RABV and V-RG, ERAGS, or Ad-0910G.

Description

Primers and probes for differentiation between Rabies virus and vaccine strains and differentiating method between Rabies virus and vaccine strains using the same}

The present invention provides a primer for discriminating Rabies virus (RABV) and Rabies virus vaccine (vaccinia rabies glycoprotein, V-RG; genetically modified rabies vaccine, ERAGS; or recombinant Adenoviruses expressing the glycoprotein of rabies, Ad-0910G) Probes and methods for differentiating rabies viruses and vaccine strains using the same.

Rabies virus is a neurophilic virus belonging to the genus Rhabdoviridae and Lyssavirus . It contains a single strand of negative RNA as a nucleic acid. Rabies virus RNA consists of five genes that express nucleoprotein (N), non-structural protein (NS), matrix protein (M), glycoprotein (G), or RNA polymerase (L). N, NS, and L proteins are involved in the formation of nucleoprotein cores, M proteins are involved in the condensation of nucleocapsids, and G proteins are involved in binding the virus to target cells.

Rabies virus is mainly infected with warm-blooded animals, causing rabies. Rabies is a common infectious disease that affects 25,000 to 75,000 people worldwide each year, with more than 29,000 cases occurring in Asia. Early symptoms of rabies include fever, headache, general weakness, anxiety, fever, boredom, and paresthesia in the bite area. Later symptoms include insomnia, anxiety, confusion, excitement, partial paralysis, hearing, excitement, saliva, Sweat, tears, excessive secretion, swallowing, water fears appear, and within a few days, delirium, convulsions, confusion, lethargy, death of respiratory muscle paralysis or complications. Rabies is transmitted by host animals such as bats, red foxes, skunks, raccoons, dogs, wolves, and mongoose.In 1993, the transmission by dogs has disappeared and has been transmitted by raccoons or badgers. Since the discovery, it has been steadily occurring and a total of 444 cases have been reported from 1993 to 2016.

In order to reduce the incidence of rabies, Korea has been continuously applying bait vaccine since 2000. Bait vaccine is an oral vaccine for the application of wild animals (fox, raccoon, coyote, etc.) that is difficult to inject into the vaccine for injection. Let's do it. Rabies are sprayed every year in mountainous areas where raccoons live, centering on rabies-prone areas in Gangwon-do and northern Gyeonggi-do, to prevent rabies from spreading.

V-RG (vaccinia rabies glycoprotein) vaccine, which is currently used as a bait vaccine, is only applied to wild raccoons and foxes due to insufficient antibody formation when orally administered to skunks, dogs, and cats. It is becoming. Thus, new rabies vaccine candidates are being studied that are safe for orphan animals (raccoons, dogs, cats) as well as non-target animals (humans, wild boars, birds, etc.). In this regard, as a rabies vaccine candidate, Korean Patent Laid-Open Publication No. 10-2015-0138956 discloses a recombinant rabies virus (ERAGS) in which 333th amino acid of rabies virus G protein is substituted with glutamic acid and 194th amino acid with serine. ), Jiyoung Choi et al ., Clin. Exp. Vaccine Res., 4: 189-194 (2015), discloses recombinant Adenoviruses expressing the glycoprotein of rabies (Ad-0910G) expressing the G protein of rabies virus.

Effective monitoring of rabies requires continuous monitoring. Direct fluorescent antibody test, mouse inoculation test, or rabies tissue culture inoculation test have been used to detect rabies virus. There is a problem of falling, and a detection method using real-time PCR has been studied. Real-time polymerase chain reaction has the advantage of less contamination during the experiment, good sensitivity and specificity, and can be quantified. In particular, multiplex real-time PCR is a method of using primer pairs that specifically bind to each of a plurality of nucleic acid molecules to be detected and probes labeled with different fluorescent dyes. Multiple different nucleic acid molecules contained in the same sample can be detected and discriminated simultaneously. Therefore, in order to monitor the effects of rabies bait vaccine, it is necessary to develop a multiplex real-time polymerase chain reaction method capable of simultaneously detecting and discriminating rabies virus and vaccine strains.

Republic of Korea Patent Publication No. 10-2015-0138956

 Jiyoung Choi et al., Clin. Exp. Vaccine Res., 4: 189-194 (2015)

An object of the present invention is rabies (Rabies virus, RABV) and rabies virus baeksinju to discriminate (vaccinia rabies glycoprotein, V-RG ;; genetically modified rabies vaccine, ERAGS or recombinant Adenoviruses expressing the glycoprotein of rabies, Ad-0910G) It is to provide a primer and probe, and a method for differentiating rabies virus and vaccine strain using the same.

In order to achieve the above object, the present invention is a first primer set comprising a forward primer consisting of a nucleotide sequence described in SEQ ID NO: 1 and a reverse primer consisting of a nucleotide sequence described in SEQ ID NO: 2, and described in SEQ ID NO: Provides a primer set that can discriminate rabies virus and V-RG (vaccinia rabies glycoprotein) vaccine strain comprising a second primer set comprising a forward primer consisting of a nucleotide sequence and a reverse primer consisting of a reverse primer consisting of a nucleotide sequence described in SEQ ID NO: 4 do.

In addition, the present invention is a forward primer consisting of a first primer set comprising a forward primer consisting of a nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of a nucleotide sequence of SEQ ID NO: 2, and a forward direction consisting of a nucleotide sequence of SEQ ID NO: 5 Provided is a primer set capable of distinguishing a rabies virus and a genetically modified rabies vaccine (ERAGS) vaccine strain comprising a third primer set comprising a primer and a reverse primer consisting of a nucleotide sequence set forth in SEQ ID NO: 6.

In addition, the present invention is a forward primer consisting of a forward primer consisting of a nucleotide sequence of SEQ ID NO: 1 and a first primer set comprising a reverse primer consisting of a nucleotide sequence of SEQ ID NO: 2, and a forward sequence consisting of the nucleotide sequence of SEQ ID NO: 7 Provided is a primer set that can discriminate between rabies virus and Ad-0910G (recombinant Adenoviruses expressing the glycoprotein of rabies) vaccine strain comprising a fourth primer set comprising a primer and a reverse primer consisting of a nucleotide sequence set forth in SEQ ID NO: 8. do.

In addition, the present invention provides a detection composition that can discriminate between rabies virus and rabies virus vaccine containing the primer set.

In addition, the present invention provides a detection kit that can discriminate between rabies virus and rabies virus vaccine comprising the composition.

In addition, the present invention is a rabies virus and rabies virus vaccine comprising the step of performing a real-time polymerase chain reaction (real-time PCR) using the primer set as a template nucleic acid isolated from the sample It provides a way to distinguish between them.

Primer and probe set of the present invention can detect rabies virus (RABV) and rabies virus vaccine strains (V-RG, ERAGS, or Ad-0910G) at the same time, and can detect the rabies virus and vaccine because of its high sensitivity. It can be useful for differentiating attention.

1 is a limit of detection (LoD) of rabies virus when performing duplex real-time PCR (DX) to discriminate between rabies virus and V-RG vaccine strain using the primer and probe set of the present invention, Amplification efficiency (Eff%), and coefficient of determination (R ² ) is confirmed.
2 is a diagram confirming the minimum detection limit, amplification efficiency, and determination coefficient of the V-RG vaccine strain when performing double real-time PCR to discriminate between rabies virus and V-RG vaccine strain using the primer and probe set of the present invention.
Figure 3 is a diagram confirming the minimum detection limit, amplification efficiency, and the coefficient of determination of rabies virus when performing dual real-time PCR to discriminate between rabies virus and ERAGS vaccine strain using the primer and probe set of the present invention.
4 is a diagram confirming the minimum detection limit, amplification efficiency, and determination coefficient of ERAGS vaccine strain when performing dual real-time PCR to discriminate between rabies virus and ERAGS vaccine strain using the primer and probe set of the present invention.
5 is a diagram confirming the minimum detection limit, amplification efficiency, and determination coefficient of rabies virus when performing double real-time PCR to discriminate between rabies virus and Ad-0910G vaccine strain using the primer and probe set of the present invention.
6 is a diagram confirming the minimum detection limit, amplification efficiency, and determination coefficient of the Ad-0910G vaccine strain when performing double real-time PCR to discriminate between rabies virus and Ad-0910G vaccine strain using the primer and probe set of the present invention.
7 is a diagram showing the detection efficiency when performing a double real-time PCR for detecting ERAGS vaccine strain using the primer and probe set of the present invention.
8 is a diagram showing the detection efficiency when performing PCR to detect ERAGS vaccine strain using a known primer set (N: control, ERAGS vaccine sample samples of 1 to 5: 10 ⁵ to 10 ¹ copies / μL concentration).
9 is a diagram showing the detection efficiency when performing PCR to detect ERAGS vaccine strain using a known primer set (N: control, ERAGS vaccine sample samples of 1 to 5: 10 ⁵ to 10 ¹ copies / μL concentration).

Hereinafter, the present invention will be described in detail.

The present invention comprises a first primer set comprising a forward primer consisting of a nucleotide sequence represented by SEQ ID NO: 1 and a reverse primer consisting of a reverse primer consisting of a nucleotide sequence represented by SEQ ID NO: 2, and a forward primer consisting of a nucleotide sequence represented by SEQ ID NO: 3; Provided is a primer set capable of discriminating rabies virus and V-RG (vaccinia rabies glycoprotein) vaccine strains comprising a second primer set comprising a reverse primer consisting of a nucleotide sequence set forth in SEQ ID NO: 4.

In addition, the present invention is a forward primer consisting of a first primer set comprising a forward primer consisting of a nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of a nucleotide sequence of SEQ ID NO: 2, and a forward direction consisting of a nucleotide sequence of SEQ ID NO: 5 Provided is a primer set capable of distinguishing a rabies virus and a genetically modified rabies vaccine (ERAGS) vaccine strain comprising a third primer set comprising a primer and a reverse primer consisting of a nucleotide sequence set forth in SEQ ID NO: 6.

In addition, the present invention is a forward primer consisting of a forward primer consisting of a nucleotide sequence of SEQ ID NO: 1 and a first primer set comprising a reverse primer consisting of a nucleotide sequence of SEQ ID NO: 2, and a forward sequence consisting of the nucleotide sequence of SEQ ID NO: 7 Provided is a primer set that can discriminate between rabies virus and Ad-0910G (recombinant Adenoviruses expressing the glycoprotein of rabies) vaccine strain comprising a fourth primer set comprising a primer and a reverse primer consisting of a nucleotide sequence set forth in SEQ ID NO: 8. do.

Primers of the primer set capable of discriminating rabies virus and V-RG vaccine strains of the present invention may be primers that specifically amplify specific regions of rabies virus or V-RG vaccine strains. In addition, the primer may be a forward or reverse primer consisting of 10 to 40, specifically 15 to 35 base sequences capable of complementarily binding to a gene of a specific region. In one embodiment of the present invention, the primer may be a forward primer consisting of a nucleotide sequence of SEQ ID NO: 1 or 3, or a reverse primer consisting of a nucleotide sequence of SEQ ID NO: 2 or 4.

Primers of the primer set capable of discriminating rabies virus and ERAGS vaccine strain of the present invention may be primers that specifically amplify specific regions of rabies virus or ERAGS vaccine strain. In addition, the primer may be a forward or reverse primer consisting of 10 to 40, specifically 15 to 35 base sequences capable of complementarily binding to a gene of a specific region. In one embodiment of the present invention, the primer may be a forward primer consisting of a nucleotide sequence of SEQ ID NO: 1 or 5, or a reverse primer consisting of a nucleotide sequence of SEQ ID NO: 2 or 6.

Primers of the primer set capable of discriminating rabies virus and Ad-0910G vaccine strain of the present invention may be primers that specifically amplify specific regions of rabies virus or Ad-0910G vaccine strain. In addition, the primer may be a forward or reverse primer consisting of 10 to 40, specifically 15 to 35 base sequences capable of complementarily binding to a gene of a specific region. In one embodiment of the present invention, the primer may be a forward primer consisting of a nucleotide sequence of SEQ ID NO: 1 or 7, or a reverse primer consisting of a nucleotide sequence of SEQ ID NO: 2 or 8.

The primers can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such primers can be modified using a number of means known in the art, as long as they exhibit the effect of detecting rabies virus or rabies virus vaccine strains. Examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages (eg, methyl phosphonates, phosphoesteres, phosphoramidates) , Carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.). Nucleic acid may be one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine , Proylene, etc.), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents. In addition, the primers of the present invention may comprise a label, if necessary, detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (eg horseradish peroxidase, alkaline phosphatase), radioisotopes (eg 32P), fluorescent molecules and chemical groups (eg biotin), and the like. have.

In addition, the present invention provides a detection composition that can discriminate between rabies virus and rabies virus vaccine containing the primer set.

The rabies virus vaccine strain may be a V-RG, ERAGS, or Ad-0910G vaccine strain.

The primer set capable of discriminating between the rabies virus and the rabies virus vaccine strain may have the characteristics as described above. In one example, the primer of the primer set may be a primer specifically amplifying a specific region of rabies virus, V-RG vaccine strain, ERAGS vaccine strain, or Ad-0910G vaccine strain, more specifically, SEQ ID NO: 1, 3, It may be a forward primer consisting of a base sequence of 5, or 7, or a reverse primer consisting of a base sequence of SEQ ID NO: 2, 4, 6, or 8.

Among the compositions, the detecting composition including the first, second, third, or fourth primer set may further include a probe consisting of a nucleotide sequence set forth in SEQ ID NO: 9, 10, 11, or 12, respectively.

As used herein, the term "probe" refers to a nucleic acid fragment corresponding to several to several hundred bases capable of specifically binding to DNA or RNA, and includes oligonucleotide probes and single stranded DNA probes. It may be prepared in the form of a double stranded DNA (double stranded DNA) probe or RNA probe.

The probe may be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such probes can be modified using many means known in the art, as long as they exhibit the effect of detecting rabies virus or rabies virus vaccine strains. Examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages (eg, methyl phosphonates, phosphoesteres, phosphoramidates) , Carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.). Nucleic acid may be one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine , Proylene, etc.), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents.

The probe may be further conjugated to a reporter at its 5 'end. The reporter is FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein (fluorescein), fluorescein chlorotriazinyl, HEX (2 ', 4', 5 ', 7'-tetrachloro -6-carboxy-4,7-dichlorofluorescein, rhodamine green, rhodamine red, tetramethylrhodamine, fluorescein isothiocyanate (FITC), oregon green, Alexa Fluorine (alexa fluor), JOE (6-Carboxy-4 ', 5'-Dichloro-2', 7'-Dimethoxyfluorescein), ROX (6-Carboxyl-X-Rhodamine), TET (Tetrachloro-Fluorescein), TRITC ( at least one selected from the group consisting of tertramethylrodamine isothiocyanate, TAMRA (6-carboxytetramethyl-rhodamine), NED (N- (1-Naphthyl) ethylenediamine), cyanine-based dyes, and thiadicarbocyanine However, in addition to the known materials that can be used as a reporter in the art, any one can be used. In one embodiment of the present invention, the reporter may be a FAM or JOE.

The probe may be further conjugated to a quencher at its 3 'end. The quencher is TAMRA, black hole quencher (BHQ) 1, BHQ2, BHQ3, nonfluorescent quencher (NFQ), dabcyl, Eclipse, deep dark quencher (DDQ), Blackberry Quencher, Iowa black It may be any one or more selected from the group consisting of), but any other known material that can be used as a quencher in the art can be used. In one embodiment of the invention, the quencher may be BHQ.

The composition may include reverse transcriptase, DNA polymerase, cofactors such as Mg 2+, dATP, dCTP, dGTP and dTTP in addition to the primer set and probe set. Various DNA polymerases can be used in the amplification step of the present invention to amplify reverse transcribed cDNA, and examples of DNA polymerases include Klenow fragments of E. coli DNA polymerase I, thermostable DNA polymerase or bacteriophage T7 DNA polymerisation. There is an enzyme. The polymerase may be isolated from the bacteria itself or purchased commercially or obtained from cells expressing high levels of the cloning gene encoding the polymerase.

In a specific embodiment of the present invention, the present inventors have a forward primer consisting of a nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7, a reverse primer consisting of a nucleotide sequence of SEQ ID NO: 2, 4, 6, or 8, And a probe consisting of the nucleotide sequence set forth in SEQ ID NO: 8. 10. 11. or 12 (see Tables 1 and 2), using this real-time polymerase chain reaction, real-time RT PCR has a limit of detection (LoD) of less than 10 copies / μL and amplification efficiency (Eff%) of rabies virus, V-RG vaccine, ERAGS vaccine, or Ad-0910G vaccine. 90% or more, the coefficient of determination (R ² ) indicating linearity is 0.995 or more (see FIGS. 1 to 6), and only rabies virus or rabies virus vaccine strain is specifically detected (see Table 5). ), Known primers It was confirmed that the detection efficiency was good when compared with the set (see FIGS. 7 to 9).

Therefore, the primer sets and probes of the present invention can be usefully used to specifically and separately detect rabies virus (RABV) and rabies virus vaccine strain (V-RG, ERAGS, or Ad-0910G).

In addition, the present invention provides a detection kit that can discriminate between rabies virus and rabies virus vaccine comprising the composition.

The rabies virus vaccine strain may be a V-RG, ERAGS, or Ad-0910G vaccine strain.

The composition may have the features as described above. In one example, the composition may comprise a primer set capable of differentiating rabies virus and rabies virus vaccine strains, wherein the primers of the primer set are specific regions of rabies virus, V-RG vaccine strain, ERAGS vaccine strain, or Ad-0910G vaccine strain. It may be a primer specifically amplifying for, more specifically, a forward primer consisting of a nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, or 7, or described as SEQ ID NO: 2, 4, 6, or 8 It may be a reverse primer consisting of a nucleotide sequence. In addition, the composition for detection, including a first, 2, 3, or 4 primer set, the composition may further comprise a probe consisting of a nucleotide sequence of SEQ ID NO: 9, 10, 11 or 12, respectively, The probe may be further conjugated to a reporter at its 5 'end and a quencher at its 3' end.

The kit may further comprise one or more other component compositions, solutions or devices suitable for the assay method. The kits include tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (in addition to specific primer pairs for specific regions of rabies virus, V-RG vaccine, ERAGS vaccine, or Ad-0910G vaccine). dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase inhibitors, RNase inhibitors, DEPC-water, sterile water and the like. On the other hand, the components included in the kit may be prepared in a liquid form, or may be prepared in a dried form in order to improve the stability of the product by lowering the degree of freedom of the included components. In order to produce such a dried form, it is necessary to apply a drying step, in which warming drying, natural drying, reduced pressure drying, freeze drying, or a combination thereof may be used.

In addition, the present invention is a rabies virus and rabies virus vaccine comprising the step of performing a real-time polymerase chain reaction (real-time PCR) using the primer set as a template nucleic acid isolated from the sample It provides a way to distinguish between them.

The rabies virus vaccine strain may be a V-RG, ERAGS, or Ad-0910G vaccine strain.

The sample may be obtained from a clinical sample or an environmental sample. For example, the clinical sample may be a sample obtained from tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, and specifically, a sample obtained from brain tissue.

The primer set capable of discriminating between the rabies virus and the rabies virus vaccine strain may have the characteristics as described above. In one example, the primer of the primer set may be a primer specifically amplifying a specific region of rabies virus, V-RG vaccine strain, ERAGS vaccine strain, or Ad-0910G vaccine strain, more specifically, SEQ ID NO: 1, 3, It may be a forward primer consisting of a base sequence of 5, or 7, or a reverse primer consisting of a base sequence of SEQ ID NO: 2, 4, 6, or 8.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 Construction of primers and probes targeting rabies virus (RABV), V-RG vaccine, ERAGS vaccine, or Ad-0910G vaccine

1-1. Primer and Probe Targeting Rabies Virus

Strains of rabies virus, which occurs mainly in Korea, were collected and collected from the US National Center for Biological Information (NCBI, https://www.ncbi.nlm.nih.gov/). Primers and probes capable of detecting rabies virus were prepared by targeting the conserved region of the collected nucleotide sequence (Table 1), and the reporter FAM (6-carboxyfluorescein) was bound to the 5 'end of the probe. The 3 'end was combined with a quencher BHQ (black hole quencher).

target Primer or probe Sequence (5 '→ 3') SEQ ID NO: RABV Forward primer GGA ATA TGT CCC GCT GAA AGA AC One Reverse primer GAG TAA CCA TTT GGG CTA CAC ACT TTA AC 2 Probe CAA GAA GAA CAT GAG GAA CTT TTG CAT CAA CG 9

1-2. Preparation of primers and probes targeting rabies virus vaccine

Primers and probes capable of detecting this by targeting rabies virus vaccine V-RG vaccine, ERAGS vaccine, or Ad-0910G vaccine were prepared (Table 2). Specifically, the US National Bioinformatics Center (NCBI, https://www.ncbi.nlm.nih.gov/) collects sequences of target genes and blasts them from NCBI to Genebank. Each registered base sequence was obtained. The obtained sequences were multi-aligned using Geneious software, and then primers and probes corresponding to the sequences appearing only in the target gene were prepared. The primer was prepared to have a melting temperature (Tm) of 60 ° C. and the probe to have a Tm of 70 ° C., and GC ratios and Tm values were calculated using prime express software. The reporter JOE (6-Carboxy-4 ', 5'-Dichloro-2', 7'-Dimethoxyfluorescein) was bound to the 5 'end of the probe, and the quencher BHQ (black hole quencher) was bound to the 3' end.

target Primer or probe Sequence (5 '→ 3') SEQ ID NO: V-RG Forward primer CGG TAA GGA AGT AGA ATC ATA AAG AAC AGT 3 Reverse primer TAA ATA GGG AAT TTC CCA AAA CAC AAT 4 Probe AGG CTC TCC TGT TTG TAC CCC TTC TGG TTT 10 ERAGS Forward primer GGT CTA CCT ACT GCT CCA CTA ACC AC 5 Reverse primer GGA TGC TCT CTT CCC TCT ACT GGA 6 Probe ATT ACA CCA TTT GGA TGC CCG AGA ATC 11 Ad-0910G Forward primer AAA AGC GGA GGT GAG ACC AGA CT 7 Reverse primer GTT AGG GAT AGG CTT ACC TTC GAA CC 8 Probe CTT GTA CAA AGT GGT TGA TCT AGA GGG CCC G 12

Experimental Example 1. Evaluation of analytical sensitivity of real-time PCR using the primer and probe set of the present invention

The analytical sensitivity of real-time PCR using the primers and probe sets prepared in Example 1 was determined from the standard curve (Limit of Detection, LoD), amplification efficiency (Eff%), and determination coefficient ( coefficient of determination (R ² ) was determined by measuring.

Specifically, the primers and probes prepared in Example 1 were mixed in the composition and concentrations of Table 3 to prepare a total of three compositions, and rabies virus, V-RG vaccine, ERAGS vaccine, or Ad- Samples of 0910G vaccine strains were obtained and quantified at concentrations of 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , 10 ¹ or 10 ⁰ copies / μL. Samples of rabies virus or V-RG vaccine strain in the RABV / V-RG duplex composition, samples of rabies virus or ERAGS vaccine strain in the RABV / ERAGS duplex composition, and samples of rabies virus or Ad-0910G vaccine strain in the RABV / Ad-0910G duplex composition, respectively. Treated. At this time, the PCR reaction solution 1 μL of the primer and probe composition diluted to a concentration of Table 3 per 1 μL, 2x RT Real-time PCR Master mix of PowerAmp ^TM One Step RT Real-time PCR Kit (Kozen Biotech, Korea) 10 μL, nuclease-free water 4 μL, and 5 μL of the virus or vaccine strain sample were mixed to bring the total volume to 20 μL. For each reaction solution, a double real-time PCR was repeated three times using the AB7500 Real-time PCR system (Life technologies, USA) under the reaction conditions described in Table 4. Using the measured cycle threshold (Ct), a standard curve was drawn and the minimum detection limit, amplification efficiency, and crystal coefficient were measured.

Composition Primer or probe Concentration (nM) RABV / V-RG duplex RABV Forward Primer 250 RABV reverse primer 250 RABV Probe 150 V-RG Forward Primer 500 V-RG reverse primer 500 V-RG Probe 200 RABV / ERAGS duplex RABV Forward Primer 500 RABV reverse primer 500 RABV Probe 150 ERAGS Forward Primer 500 ERAGS reverse primer 500 ERAGS probe 300 RABV / Ad-0910G duplex RABV Forward Primer 500 RABV reverse primer 500 RABV Probe 200 Ad-0910G Forward Primer 500 Ad-0910G Reverse Primer 500 Ad-0910G Probe 200

Temperature (℃) time Number of cycles 50 30 minutes One 95 10 minutes One 95 15 seconds 40 60 1 minute

As a result, as shown in Figures 1 to 6, the minimum detection limit of real-time PCR using the primer and probe set of the present invention is 10 copies / μL of rabies virus, 1 copy / μL of V-RG vaccine, 1 copy / μL of ERAGS vaccine , And Ad-0910G vaccine strains were measured at 10 copies / μL, amplification efficiency of 90% or higher, and crystal coefficient of 0.995 or higher. From this, it was found that the rabies virus and vaccine strain can be detected with high sensitivity using each composition of Table 3.

Experimental Example 2. Evaluation of specificity of real-time PCR using the primer and probe set of the present invention

Analytical specificity of real-time PCR using primers and probe sets prepared in Example 1 was confirmed by cross-reaction with other strains.

Specifically, in addition to rabies virus, V-RG vaccine strain, ERAGS vaccine strain, and Ad-0910G vaccine strain, 29 strains of the pathogen (Table 5) gene were used to confirm cross-reaction by other strains. Specifically, the same as those described in Experiment 1 except that the pathogen gene gDNA or gRNA of Table 5 was used at a concentration of 10 ng / μL instead of the rabies virus, V-RG vaccine line, ERAGS vaccine line, or Ad-0910G vaccine line gene. Real time PCR was performed by the method.

number category Strain name RABV / V-RG duplex RABV / ERAGS duplex RABV / Ad-0910G duplex One Rabies virus Rabies virus + - + - + - 2 Rabies virus vaccine strain V-RG strain - + - - - - 3 ERAGS strain + - + + + - 4 Ad-0910G strain - - - - - + 5 Influenza virus Influenza A H1 - - - - - - 6 Influenza A H3 - - - - - - 7 Influenza A H5 - - - - - - 8 Respiratory virus Adenovirus - - - - - - 9 Parainfluenza 1 - - - - - - 10 Parainfluenza 2 - - - - - - 11 Parainfluenza 3 - - - - - 12 Coronavirus - - - - - - 13 Respiratory syncytial virus A - - - - - - 14 Respiratory syncytial virus B - - - - - - 15 Measles - - - - - - 16 Rubella - - - - - - 17 Mumps - - - - - - 18 Pathogenic bacteria Bacillus cereus - - - - - - 19 Bacillus subtilis - - - - - - 20 Campylobacter jejuni - - - - - - 21 E.coli O157 - - - - - - 22 Listeria monocytogenes - - - - - - 23 Yersinia enterocolitica - - - - - - 24 Clostridium perfringens - - - - - - 25 Salmonella typhi - - - - - - 26 Salmonella typhimurium - - - - - - 27 Shigella sonnei - - - - - - 28 Staphylococcus aureus - - - - - - 29 Vibrio vulnificus - - - - - - 30 Yersinia enterocolitica - - - - - - 31 Legionella pneumophila - - - - - - 32 Pseudomonas aeruginosa - - - - - - 33 Mycoplasma pneumoniae - - - - - - 34 Other species Cow - - - - - - 35 Pork - - - - - - 36 Chicken - - - - - - 37 Duck - - - - - - 38 Sheep - - - - - - 39 Goat - - - - - - 40 Turkey - - - - - - 41 Raccoon dog - - - - - - 42 Horse - - - - - - 43 Donkey - - - - - - 44 Human - - - - - - 45 Dog - - - - - -

As a result, as shown in Table 5, the RABV / V-RG duplex composition of the present invention is a rabies virus and V-RG vaccine strain, the RABV / ERAGS duplex composition is a rabies virus and ERAGS vaccine strain, RABV / Ad-0910G duplex composition Was separately detected from rabies virus and Ad-0910G vaccine strain. From this, it can be seen that the primer and probe set of the present invention do not exhibit cross-reaction by strains other than rabies virus and vaccine strain.

Experimental Example 3. Confirmation of detection efficiency of real-time PCR using the primer and probe set of the present invention

In order to confirm the superiority of the analytical sensitivity of real-time PCR using the primer and probe set of the present invention, the primer set for discrimination of known RABV and ERAGS vaccine strains (Korean Patent Publication No. 10-2015-0138956; known primer set 1) and Real-time PCR analysis was performed in the same manner as in Experimental Example 1 using a primer set for detecting RABV (a standard for diagnosing etiological diseases, Agricultural and Livestock Quarantine Headquarters, 2015; known primer set 2).

Specifically, primers and probe sets targeting rabies virus and ERAGS vaccine strain prepared in Example 1 and two known primer sets (Table 6) were prepared, and ERAGS vaccine strain samples were sold by the Korean Veterinary Gene Bank. Taken and quantified at a concentration of 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , or 10 ¹ copies / μL. At this time, PCR reaction solution by a diluted primer such that 50 pmole / μL for a similar manner to that described in Experimental Example 1 for the primer and probe set of the present invention, known primer set 1, each 1 μL, PowerAmp ^TM One Mix 12.5 μL of 2x RT Real-time PCR Master mix from Step RT Real-time PCR Kit (Kogen Biotech, Korea), 3.5 μL of nuclease-free water, and 5 μL of ERAGS vaccine stock sample to a total volume of 25 μL. for the second set by each 1 μL of the primer diluted to ^{10 pmole / μL, PowerAmp TM one} Step RT Real-time PCR Kit ( kojen Biotech, Korea) of 2x RT Real-time PCR Master mix 10 μL, nuclease- 3 μL of free water and 5 μL of the ERAGS vaccine strain sample were mixed to bring the total volume to 20 μL. The primers and prop sets of the present invention were subjected to double real time PCR in the same manner as described in Experimental Example 1, and the PCR was performed using the GeneAmp 9700 PCR System (Life technologies, USA) for the known primer sets 1 and 2. 5 μL of PCR product was then electrophoresed on agarose gel.

division target Primer or probe Sequence (5 '→ 3') SEQ ID NO: Primer and Probe Set of the Invention RABV Forward primer GGA ATA TGT CCC GCT GAA AGA AC One Reverse primer GAG TAA CCA TTT GGG CTA CAC ACT TTA AC 2 Probe CAA GAA GAA CAT GAG GAA CTT TTG CAT CAA CG 9 ERAGS Forward primer GGT CTA CCT ACT GCT CCA CTA ACC AC 5 Reverse primer GGA TGC TCT CTT CCC TCT ACT GGA 6 Probe ATT ACA CCA TTT GGA TGC CCG AGA ATC 11 Notice Primer Set 1 RABV Forward primer AGG RAA TTG GGC TTT GAC TGG A 13 Reverse primer AAA GGG GCT GTC TCG AAA AT 14 ERAGS Forward primer TGT CTT GTG ACA TTT TTA CCT CC 15 Reverse primer GGA TGA TCT CAT TCC AAG TTT C 16 Notice Primer Set 2 RABV Forward primer GCA GAT AGG ATA GAG CAR A 17 Reverse primer AAA GTG AAT GAG ATT GAA C 18

As a result, as shown in Figs. 7 to 9, the ERAGS vaccine strain was detected by the RABV primer and probe set in the primer and probe set of the present invention, and the concentration of 10 copies / μL by the ERAGS primer and probe set, and known primer set. From 1 to 10 ³ copies / μL by the RABV primer set, up to 10 ⁴ copies / μL by the ERAGS primer set, up to 10 copies / μL by the RABV primer set from known primer set 2 It became. The primers and probe sets of the present invention detected lower concentrations of the primers and probe sets of the present invention than the known primer sets 1, and the primers and probe sets of the present invention had the same detectable concentrations as the primers and probe sets of the present invention, but the RABV and ERAGS vaccine strains Was not detected separately. From this, it can be seen that the RABV and ERAGS vaccine strains can be detected with high efficiency when using the primers and probe sets of the present invention compared to known primers.

<110> KoGene BioTech Co., LTD. <120> Primers and probes for differentiation between Rabies virus and          vaccine strains and differentiating method between Rabies virus          and vaccine strains using the same <130> 2018P-07-045 <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RABV Forward Primer <400> 1 ggaatatgtc ccgctgaaag aac 23 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> RABV Reverse Primer <400> 2 gagtaaccat ttgggctaca cactttaac 29 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> V-RG Forward Primer <400> 3 cggtaaggaa gtagaatcat aaagaacagt 30 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> V-RG Reverse Primer <400> 4 taaataggga atttcccaaa acacaat 27 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> ERAGS Forward Primer <400> 5 ggtctaccta ctgctccact aaccac 26 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ERAGS Reverse Primer <400> 6 ggatgctctc ttccctctac tgga 24 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Ad-0910G Forward Primer <400> 7 aaaagcggag gtgagaccag act 23 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Ad-0910G Reverse Primer <400> 8 gttagggata ggcttacctt cgaacc 26 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> RABV Probe <400> 9 caagaagaac atgaggaact tttgcatcaa cg 32 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> V-RG Probe <400> 10 aggctctcct gtttgtaccc cttctggttt 30 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> ERAGS Probe <400> 11 attacaccat ttggatgccc gagaatc 27 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Ad-0910G Probe <400> 12 cttgtacaaa gtggttgatc tagagggccc g 31 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RABV Forward Primer_known 1 <400> 13 aggraattgg gctttgactg ga 22 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RABV Reverse Primer_known 1 <400> 14 aaaggggctg tctcgaaaat 20 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ERAGS Forward Primer_known 1 <400> 15 tgtcttgtga catttttacc tcc 23 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ERAGS Reverse Primer_known 1 <400> 16 ggatgatctc attccaagtt tc 22 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RABV Forward Primer_known 2 <400> 17 gcagatagga tagagcara 19 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RABV Reverse Primer_known 2 <400> 18 aaagtgaatg agattgaac 19

Claims (10)

A first primer set comprising a forward primer consisting of a nucleotide sequence represented by SEQ ID NO: 1 and a reverse primer consisting of a reverse primer consisting of a nucleotide sequence represented by SEQ ID NO: 2, and a forward primer consisting of the nucleotide sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4 A primer set capable of discriminating rabies virus and V-RG (vaccinia rabies glycoprotein) vaccine strains comprising a second primer set comprising a reverse primer consisting of a nucleotide sequence as described below.
A first primer set comprising a forward primer consisting of a nucleotide sequence represented by SEQ ID NO: 1 and a reverse primer consisting of a reverse primer consisting of a nucleotide sequence represented by SEQ ID NO: 2, and a forward primer consisting of the nucleotide sequence represented by SEQ ID NO: 5 and SEQ ID NO: 6 A primer set that can discriminate between a rabies virus and a genetically modified rabies vaccine (ERAGS) vaccine strain, including a third primer set comprising a reverse primer consisting of a nucleotide sequence as described below.
A first primer set comprising a forward primer consisting of a nucleotide sequence represented by SEQ ID NO: 1 and a reverse primer consisting of a reverse primer consisting of a nucleotide sequence represented by SEQ ID NO: 2, and a forward primer consisting of the nucleotide sequence represented by SEQ ID NO: 7 and SEQ ID NO: 8 A primer set that can discriminate between a rabies virus and Ad-0910G (recombinant Adenoviruses expressing the glycoprotein of rabies) vaccine strain, including a fourth primer set comprising a reverse primer consisting of a nucleotide sequence as described below.
The detection composition which can discriminate between rabies virus and rabies virus vaccine containing the primer set of Claim 1, 2, or 3.
The detection composition according to claim 4, wherein the detection composition comprising the primer set of claim 1 further comprises a rabies virus and a rabies virus vaccine strain further comprising a probe consisting of a nucleotide sequence of SEQ ID NOs: 9 and 10, respectively. .
The detection composition according to claim 4, wherein the detection composition comprising the primer set of claim 2 is capable of discriminating rabies virus and rabies virus vaccine strain further comprising a probe consisting of base sequences described in SEQ ID NOs: 9 and 11, respectively. .
The detection composition according to claim 4, wherein the composition for detection comprising the primer set of claim 3 is capable of discriminating rabies virus and rabies virus vaccine strain further comprising a probe consisting of base sequences described in SEQ ID NOs: 9 and 12, respectively. .
The detection kit which can discriminate between rabies virus and rabies virus vaccine containing the composition of Claim 4.
Performing a real-time polymerase chain reaction (real-time PCR) using the primer set of claim 1, 2 or 3 with the nucleic acid isolated from the sample as a template. How to differentiate between rabies virus and rabies virus vaccine.
The method of claim 9, wherein the sample is capable of discriminating rabies virus and rabies virus vaccine strains obtained from clinical or environmental samples.

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