KR102284243B1 - Primers and probes for simultaneous detection of nipah and hendra viruses and detecting method for nipah and hendra viruses using the same - Google Patents

Abstract

The present invention relates to a primer and probe for simultaneously detecting Nipah virus and Hendra virus, and a method for detecting Nipah virus and Hendra virus using the same. More specifically, the primer and probe capable of simultaneously detecting Nipa virus and Hendra virus have excellent sensitivity and can specifically detect Nipa virus and Hendra virus. Accordingly, a set of primer and probe according to the present invention can be useful for detection and diagnosis of Nipa virus and Hendra virus and can be helpful to develop effective preventive measures for the viruses.

Description

Primers and probes for simultaneously detecting Nipah and Hendra viruses, and a method for detecting Nipah and Hendra viruses using the same

The present invention relates to a primer and a probe for simultaneously detecting Nipah virus and Hendra virus, and a method for detecting Nipah virus and Hendra virus using the same.

Nipah virus was isolated from adults with respiratory symptoms in Malaysia and Singapore in 1999 and was named Nipah after the area where it was first isolated. Hendra virus was first discovered in 1994 in horses and humans raised in a stable in Hendra, Australia, and was named Hendra after the area.

Nipah virus and Hendra virus have negative-strand RNA of 18.2 kb as a gene, and this gene includes nucleocapsid (N), phosphoprotein (P), matrix (M), fusion protein ( fusion, F), attachment glycoprotein (G), and large polymerase (L). The two viruses belong to the Paramyxoviridae family, and the two viruses are closely related genetically and immunologically. Like other paramyxoviridae viruses, both viruses are infected by attachment of an adhesion protein (G) and a fusion protein (F) to the receptor of the host cell. B2, -B3) is known to be attached to receptors.

Both Nipah virus and Hendra virus have characteristics such as high fever, headache, dizziness, sore throat, and narcolepsy, and interstitial pneumonia is characteristic of patients infected with Hendra virus. Nipah and Hendra viruses are zoonotic viruses and when they occur in animals, most infected animals recover to normal, but in humans, they cause severe encephalitis-like symptoms and have a very high mortality rate.

The regions where Nipah and Hendra virus infection mainly occur are relatively poor countries in tropical and subtropical regions, and in developed countries, research on Nipah and Hendra virus diagnosis methods is insufficient because they enter the country after infection rather than within the country. .

In foreign countries, so far, 105 out of 265 patients have died in Malaysia, 97 out of 135 patients in Bangladesh, and 17 out of 19 patients in India, showing a high fatality rate. There has never been an outbreak of Hendra virus infection.

Although there has been no domestic introduction of Nipah and Hendra viruses so far, such as avian influenza (1997, Hong Kong), SARS (2003, China), MERS (2015, Korea), and most recently Corona 19 virus, There is concern, and since Nipah and Hendra viruses have a very high fatality rate, it is a virus that has not yet been introduced and has no history of infection in Korea. need.

Accordingly, research related to methods for detecting Nipah and Hendra viruses has continued, and it is possible to detect Nipah or Hendra viruses using reverse transcription polymerase chain reaction (RT-PCR), real-time PCR, etc. A method, a composition for detection, and a kit including the composition have been developed. However, conventional methods for detecting Nipah and Hendra viruses using reverse transcription polymerase chain reaction (RT-PCR) have limitations in specificity and sensitivity to Nipah and Hendra viruses.

The present inventors set out to develop a new detection method for Nipah and Hendra viruses, which has a better effect than the existing detection method, and as a result, it is possible to simultaneously detect and confirm both viruses by targeting the M gene of Nipah and Hendra viruses. By making a primer and a probe set and performing a real-time multiplex gene amplification technique using the primer and probe set, it was confirmed that Nipa and Hendra viruses can be simultaneously specifically detected with excellent sensitivity quickly, thereby completing the present invention did.

It is an object of the present invention to provide a method for detecting Nipah virus and Hendra virus for preemptive response to and monitoring the influx of Nipah virus and Hendra virus, which exhibit a high fatality rate, and construction of an inspection system for a pandemic.

In order to achieve the above object, the present invention provides a primer set capable of simultaneously detecting Nipa and Hendra viruses including all nucleotide sequences shown in SEQ ID NOs: 1, 2, 4 and 5, respectively.

In addition, the present invention provides a probe set capable of simultaneously detecting Nipa and Hendra viruses including all of the nucleotide sequences shown in SEQ ID NOs: 3 and 6, respectively.

In addition, the present invention provides a composition for detection capable of simultaneously detecting Nipa and Hendra viruses including the primer set and the probe set according to the present invention.

In addition, the present invention provides a detection kit capable of simultaneously detecting Nipa and Hendra viruses comprising the composition of the present invention.

In addition, the present invention provides a method for simultaneously detecting Nipa and Hendra viruses, which includes performing a real-time polymerase chain reaction using a sample as a template and a primer set and a probe set according to the present invention.

The primer and probe set of the present invention can specifically and simultaneously detect Nipah and Hendra viruses, and have excellent detection efficiency, such as analytical sensitivity, compared to known primer sets. and can be helpful in preparing effective quarantine measures against the virus.

1 is a diagram confirming that only Nipah virus is detected by performing gene multiple alignment with Hendra virus using a primer and probe set targeting Nipah virus.
2 is a diagram confirming that only Hendra virus is detected by performing gene multiple alignment with Nipah virus using a primer and probe set targeting Hendra virus.
3 is a diagram comparing the experimental results with a single real-time polymerase chain reaction in order to confirm the presence or absence of inhibition/competition of the multiple real-time polymerase chain reaction of the present invention.
Figure 3a is a graph of the experimental results when the Nipah virus single system treated with only the Nipah virus template alone.
Figure 3b is a graph of the experimental results when only the Hendra virus template was treated alone in the Hendra virus single system.
Figure 3c is a graph of the experimental results in the case of Nipah / Hendra virus multi system treated with only the Nipah virus template alone.
Figure 3d is a graph of experimental results in the case of treating only the Hendra virus template alone in the Nipa / Hendra virus multi system.
Figure 3e is a graph of the experimental results when the Nipa / Hendra virus multi system was treated with the Nipa / Hendra virus template at the same time.
4 is a graph confirming whether the experimental results are different depending on whether or not the internal control (IC) is added to the multiple real-time polymerase chain reaction system for detecting Nipa and Hendra viruses.
Figure 4a is a graph of the experimental results when the internal control (IC) was not added to the multi-real-time polymerase chain reaction system for detecting Nipa and Hendra viruses.
Figure 4b is a graph of experimental results when an internal control (IC) is added to a multi-real-time polymerase chain reaction system for detecting Nipa and Hendra viruses.
5 is a graph showing the results of analyzing the Nipah virus detection limit of the primer and probe set of the present invention according to the existing real-time polymerase chain reaction conditions and high-speed real-time polymerase chain reaction conditions.
Figure 5a is a graph of the analysis result of the Nipah virus detection limit of the primer and probe set of the present invention under the existing real-time polymerase chain reaction conditions.
Figure 5b is a graph showing the results of analyzing the Nipah virus detection limit of the primer and probe set of the present invention under high-speed real-time polymerase chain reaction conditions.
6 is a graph showing the results of analyzing the Hendra virus detection limit of the primer and probe set of the present invention according to the existing real-time polymerase chain reaction conditions and high-speed real-time polymerase chain reaction conditions.
Figure 6a is a graph of the analysis result of the Hendra virus detection limit of the primer and probe set of the present invention under the existing real-time polymerase chain reaction conditions.
6b is a graph showing the results of analyzing the Hendra virus detection limit of the primer and probe set of the present invention under high-speed real-time polymerase chain reaction conditions.
7 is a graph showing the result of analyzing the detection limit of the Nipah virus-specific detection system using the primer and probe set of the present invention.
Figure 7a is a log graph of the amplification curve of the real-time polymerase chain reaction using the primer and probe set of the present invention for the purpose of specific detection of Nipah virus.
7B is a graph of a standard curve of a real-time polymerase chain reaction using the primer and probe set of the present invention for the purpose of specific detection of Nipah virus.
8 is a graph showing the result of analyzing the detection limit of the Hendra virus-specific detection system using the primer and probe set of the present invention.
8A is a log graph of an amplification curve of a real-time polymerase chain reaction using the primer and probe set of the present invention for the purpose of specific detection of Hendra virus.
8B is a graph of a standard curve of a real-time polymerase chain reaction using the primer and probe set of the present invention for the purpose of specific detection of Hendra virus.

Hereinafter, the present invention will be described in detail.

The present invention provides a primer set capable of simultaneously detecting Nipa and Hendra viruses including all of the nucleotide sequences shown in SEQ ID NOs: 1, 2, 4 and 5, respectively.

The primers comprising the nucleotide sequences respectively shown in SEQ ID NOs: 1 and 2 are primers for detecting Nipah virus. The primers including the nucleotide sequences respectively shown in SEQ ID NOs: 4 and 5 are primers for detecting Hendra virus.

The primer set is a primer set that specifically amplifies the M gene region of Nipah virus and the M gene region of Hendra virus. In addition, the primer set is a pair of forward and reverse primers comprising 10 to 40, specifically 20 to 30 nucleotide sequences capable of complementary binding to the gene of the M gene region of Nipah virus or the M gene region of Hendra virus. . In one embodiment of the present invention, each of the base sequences described in SEQ ID NOs: 1, 2, 4 and 5 is included.

The primer can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. These primers can be modified using many means known in the art as long as they exhibit an effect capable of detecting Nipah virus or Hendra virus. Examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (eg, methyl phosphonates, phosphotriesters, phosphoroamidates). , carbamates, etc.) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.). Nucleic acids may contain one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (eg, acridine). , proralene, etc.), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents. In addition, if necessary, the primer of the present invention may include a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (eg, horseradish peroxidase, alkaline phosphatase), radioactive isotopes (eg, ³² P), fluorescent molecules and chemical groups (eg, biotin), and the like. There is this.

In addition, the present invention provides a probe set capable of simultaneously detecting Nipa and Hendra viruses including all of the nucleotide sequences shown in SEQ ID NOs: 3 and 6, respectively.

The probe including the nucleotide sequence shown in SEQ ID NO: 3 is a probe for detecting Nipah virus. The probe including the nucleotide sequence shown in SEQ ID NO: 6 is a Hendra virus detection probe.

As used herein, the term “probe” refers to a nucleic acid fragment corresponding to several to hundreds of bases capable of specifically binding to DNA or RNA, an oligonucleotide probe, a single stranded DNA probe , a double-stranded DNA probe or an RNA probe may be prepared.

The probe can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such a probe can be modified using many means known in the art as long as it exhibits an effect capable of detecting Nipah virus or Hendra virus. Examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (eg, methyl phosphonates, phosphotriesters, phosphoroamidates). , carbamates, etc.) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.). Nucleic acids may contain one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (eg, acridine). , proralene, etc.), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents.

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

The probe is further conjugated with a quencher at its 3' end. The quencher is TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dabcyl, Eclipse, DDQ (Deep Dark Quencher), Blackberry Quencher (Blackberry Quencher), may be any one or more selected from the group consisting of Iowa black (Iowa black), in addition, any known material that can be used as a quencher in the art may be used. In one embodiment of the invention, the quencher is BHQ1.

In addition, the present invention provides a primer set including all nucleotide sequences set forth in SEQ ID NOs: 1, 2, 4 and 5, respectively, and Nipa and Hendra including a probe set including all base sequences set forth in SEQ ID NOs: 3 and 6, respectively A composition for detection capable of simultaneously detecting a virus is provided.

The primer set has the same characteristics as described above. For example, the primer set may be a primer set that specifically amplifies the M gene region of Nipah virus and the M gene region of Hendra virus. In addition, the primer set is a pair of forward and reverse primers comprising 10 to 40, specifically 20 to 30 nucleotide sequences capable of complementary binding to the gene of the M gene region of Nipah virus or the M gene region of Hendra virus. . In one embodiment of the present invention, each of the base sequences described in SEQ ID NOs: 1, 2, 4 and 5 is included.

The probe set has the characteristics as described above. In one example, the probe set is further conjugated with a reporter at its 5' end and a quencher at its 3' end. In one embodiment of the present invention, the reporter is FAM or HEX, and the quencher is BHQ1.

The composition may include reverse transcription polymerase, DNA polymerase, ^{cofactors such as Mg 2+} , dATP, dCTP, dGTP and dTTP in addition to the primer set and probe set. In order to amplify the reverse transcribed cDNA, various DNA polymerases may be used in the amplification step of the present invention, and examples of the DNA polymerase include Klenow fragment of E. coli DNA polymerase I, thermostable DNA polymerase or bacteriophage T7 DNA polymerization. There are enzymes. Polymerases can be isolated from the bacterium itself, purchased commercially, or obtained from cells expressing high levels of cloning genes encoding polymerases.

In a specific example of the present invention, the present inventors prepared primers including the nucleotide sequences shown in SEQ ID NOs: 1, 2, 4 and 5, respectively, and probes including the nucleotide sequences shown in SEQ ID NOs: 3 and 6, respectively. (See Figures 1, 2 and Table 3).

In addition, the present inventors have developed a multiplex real-time PCR assay for detecting Nipah and Hendra viruses (see FIGS. 3, 4 and 4), and the concentrations and experimental conditions of primers and probes. Optimized (see Tables 5 and 6), and fast real-time PCR conditions were established (see FIGS. 5, 6, Tables 8, 9 and 10).

In addition, as a result of performing multiplex real-time RT-PCR using the primer and probe set, the present inventors confirmed that the detection limit for each of Nipa and Hendra viruses was 1.0×10 ³ copies/μl or less, and specifically confirmed that it can be detected even when the RNA of the M gene of Nipa and Hendra viruses is ^{diluted to 1.0×10 2} copies/μl (see Table 11, FIGS. 7 and 8), and the viruses are specifically distinguished and detected was confirmed (see Table 12).

Therefore, the primer and probe set can be usefully used for detection and diagnosis of Nipa and Hendra viruses.

In addition, the present invention provides a detection kit capable of simultaneously detecting Nipah and Hendra viruses comprising the composition for detection capable of simultaneously detecting Nipah and Hendra viruses of the present invention.

The composition has the characteristics as described above. In one example, the composition includes a primer set including all nucleotide sequences set forth in SEQ ID NOs: 1, 2, 4 and 5, respectively, and a probe set including all base sequences set forth in SEQ ID NOs: 3 and 6, respectively. The primer set is a primer set that specifically amplifies the M gene region of Nipah virus and the M gene region of Hendra virus. In one embodiment of the present invention, the primer set includes the nucleotide sequences described in SEQ ID NOs: 1, 2, 4 and 5, respectively. The probe set is further conjugated with a reporter at its 5' end and a quencher at its 3' end. In one embodiment of the present invention, the reporter is FAM or HEX, and the quencher is BHQ1.

The kit may further include one or more other component compositions, solutions or devices suitable for the assay method. In addition to a pair of primers specific for the M gene region of Nipah virus and the M gene region of Hendra virus, the kit includes a tube or other suitable container, reaction buffer (varying pH and magnesium concentration), deoxynucleotides (dNTPs), Hot start Taq -Enzymes such as polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC-water and 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 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, and in this case, heating drying, natural drying, reduced pressure drying, freeze drying, or a combination process thereof may be used.

In addition, the present invention uses a nucleic acid isolated from a sample as a template, and a primer set including all nucleotide sequences described in SEQ ID NOs: 1, 2, 4 and 5, respectively, and all nucleotide sequences described in SEQ ID NOs: 3 and 6, respectively. Provided is a method for simultaneously detecting Nipa and Hendra viruses, comprising performing a real-time polymerase chain reaction using a probe set.

In the method, the real-time polymerase chain reaction may be a high-speed real-time polymerase chain reaction. For example, while the reaction time of the conventional real-time polymerase chain reaction is 2 hours, the high-speed real-time polymerase chain reaction minimizes the reverse transcription process and the annealing/extension time under the existing reaction conditions. Thus, the reaction time can be shortened to 80 minutes.

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.

The primer set and the probe set may have the same characteristics as described above. In one example, the primer set specifically amplifies the M gene region of Nipah virus and the M gene region of Hendra virus. In addition, the primer set is a pair of forward and reverse primers comprising 10 to 40, specifically 20 to 30 nucleotide sequences capable of complementary binding to the gene of the specific region. In addition, the probe set is further conjugated with a reporter at its 5' end and a quencher at its 3' end. In one embodiment of the present invention, the reporter is FAM or HEX, and the quencher is BHQ1.

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

However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited thereto.

<Example 1> Preparation of primers and probes for detecting Nipa and Hendra viruses

After searching for the gene sequences for Nipah and Hendra viruses in the National Center for Biological Information (NCBI, https://www.ncbi.nlm.nih.gov/) database, each sequence was arranged, A nucleotide sequence database of 17 Hendra virus was obtained (Tables 1 and 2). Multi-alignment was performed using the nucleotide sequence for each virus, and primers and probes were designed and manufactured in a conserved region ( FIGS. 1 , 2 and 3 ). The probes (SEQ ID NOs: 3 and 6) designed in this study showed 100% detection rates for each of Nipah virus and Hendra virus when sequence alignment was performed on the entire collected database.

List of nucleotide sequence names of 15 types of Nipah virus Registration Number sequence name AF212302 Nipah virus, complete genome AF376747 Nipah virus nucleocapsid protein (N), V protein (P/V/C), phosphoprotein (P/V/C), C protein (P/V/C), matrix protein (M), fusion protein (F), and glycoprotein (G) genes, complete cds AJ564621 Nipah virus complete genome, isolate NV/MY/99/VRI-2794 AJ564622 Nipah virus complete genome, isolate NV/MY/99/VRI-1413 AJ564623 Nipah virus complete genome, isolate NV/MY/99/UM-0128 AJ627196 Nipah virus complete genome, isolate NV/MY/99/VRI-0626 AY029767 Nipah virus isolate UMMC1, complete genome AY029768 Nipah virus isolate UMMC2, complete genome AY988601 Nipah virus from Bangladesh, complete genome FJ513078 Nipah virus isolate Ind-Nipah-07-FG from India, complete genome FN869553 Nipah virus N gene, P gene, M gene, F gene, G gene and L gene, isolated from urine of Pteropus vampyrus, genomic RNA JN808857 Nipah virus isolate NIVBGD2008MANIKGONJ, complete genome JN808864 Nipah virus isolate NIVBGD2010FARIDPUR, partial genome KY425646 Nipah virus isolate IRF0160, partial genome KY425655 Nipah virus isolate IRF0158, partial genome

List of nucleotide sequence names of 17 types of Hendra virus Registration Number sequence name AF017149 Hendra virus, complete genome HM044317 Hendra virus strain HeV/Australia/Horse/2008/Redlands, complete genome HM044318 Hendra virus strain HeV/Australia/Horse/2006/Murwillumbah, complete genome HM044319 Hendra virus strain HeV/Australia/Horse/2007/Peachester, complete genome HM044320 Hendra virus strain HeV/Australia/Horse/2008/Proserpine, complete genome HM044321 Hendra virus strain HeV/Australia/Horse/2007/CliftonBeach, complete genome JN255800 Hendra virus strain HeV/Australia/Bat/2009/Yeppoon Y47a, complete genome JN255803 Hendra virus strain HeV/Australia/Bat/2009/CedarGrove 11c, complete genome JN255804 Hendra virus strain HeV/Australia/Horse/2007/Clifton Beach Q, complete genome JN255806 Hendra virus strain HeV/Australia/Horse/2009/Cawarral, complete genome JN255812 Hendra virus strain HeV/Australia/Horse/2010/Bowen, partial genome JN255813 Hendra virus strain HeV/Australia/Horse/2007/Peachester, partial genome JN255814 Hendra virus strain HeV/Australia/Bat/2008/Gordonvale, partial genome JN255815 Hendra virus strain HeV/Australia/Horse/2008/Proserpine, partial genome JN255817 Hendra virus strain HeV/Australia/Human Male/2008/Redlands, partial genome JN255818 Hendra virus strain HeV/Australia/Human/2009/Cawarral, partial genome KY425627 Hendra virus isolate IRF0167, partial genome

Targets Nipah and Hendra viruses Primer and probe sequences virus Primer or probe ^a sequence (5'→3') target SEQ ID NO: nepah
virus Nipah-M-F AGG ACA ATT GCT GCC TAC CCT M gene One Nipah-M-R ATT TTC TCA GTT GAT CCA GCT GTT CT 2 Nipah-M-Probe (FAM, BHQ1) ACT TTG AGG GAA CAG AGT TCC TCC AGA AGA TC 3 Hendra
virus Hendra-M-F CAT TGC CGC GTA CCC TCT T M gene 4 Hendra-M-R TGT GAC TTT CAA AGA ACA TAG CTC TTC 5 Hendra-M-Probe (HEX, BHQ1) TTG GCA AGA GCA CTT CTC ACC CTC AGG 6

^a F: forward primer

R: reverse primer

<Example 2> Development and optimization of multiplex real-time PCR assay for detecting Nipah and Hendra viruses

<2-1> Development of multiplex real-time PCR test method

In the present invention, a polymerase chain reaction was performed using a real-time PCR device (Applied Biosystems 7500 Real-Time PCR System (ThermoFisher)) to detect Nipah and Hendra viruses. In addition, the performance of the multiplex real-time PCR system was confirmed by diluting the prepared positive sample ( in vitro transcribed RNA).

Specifically, the single-plex real-time PCR for Nipah virus and Hendra virus whether inhibition/competition in the configuration of multiplex real-time PCR ) system and a multiplex real-time PCR system were used as a single positive control and a multi positive control for each virus system, and the template used for each (template) was used by serial dilution ^{from 1.0×10 6} to 1.0×10 ^{0 copies/μl.}

In addition, in order to add an internal control (IC) that can check the presence of inhibitors in the sample and whether the reaction reagent is normal, an inhibition test according to the addition of IC was performed.

As a result, it was confirmed that both multiplex (multi-plex) and single-plex (single-plex) were ^{detected up to 1.0×10 1} copies/μl regardless of dilution conditions (FIG. 3). In addition, as a result of the inhibition test, it was confirmed that the presence or absence of IC did not affect the amplification of Nipa and Hendra viruses (Table 4 and FIG. 4). This suggests that, in the case of the multiple real-time polymerase chain reaction of the present invention, even when two or more targets are simultaneously detected, inhibition/competition does not occur, and thus the detection performance for Nipa and Hendra viruses is not reduced.

Results before and after IC addition of Nipa and Hendra virus multiplex real-time polymerase chain reaction system concentration of template
(copies/μl) Nipah virus (Ct value) Hendra virus (Ct value) Without IC When there is an IC Without IC When there is an IC 1.0×10 ⁶ 19.5 19.23 19.32 19.26 1.0×10 ⁵ 22.86 22.64 22.78 22.63 1.0×10 ⁴ 26.07 25.8 26.24 25.87 1.0×10 ³ 29.32 29.12 29.45 29.26 1.0×10 ² 32.67 32.61 32.75 32.53 1.0×10 ¹ 35.92 35.34 36.72 25.4 1.0×10 ⁰ Nd (0/2) 37.7 (2/2) 39.1 (1/2) 39.26 (1/2)

<2-2> Optimization of primer and probe concentrations and experimental conditions

Through various optimization processes, the concentrations and experimental conditions of primers and probes of the multiplex real-time polymerase chain reaction for detection of Nipa and Hendra viruses were optimized and confirmed (Tables 5 and 6). After diluting the primers and probes used for detecting Nipah and Hendra virus to the respective concentrations used, dilute the diluted primer/probe mix, AgPath-ID™ One-Step RT-PCR Reagents and template to prepare a PCR purification kit. qPCR was performed (Table 7). And, the basic performance evaluation of multiple real-time polymerase chain reaction was verified with the prepared positive sample (in vitro transcribed RNA). In addition, the accuracy of the analysis result was improved by including the IC for PCR inhibition and quality control (QC) of the analysis result.

Multiple real-time PCR reaction conditions Temperature (℃) time cycle 50 30 minutes One 95 10 minutes One 95 15 seconds 45
60 1 min

Final concentrations of primers and probes primer or probe ^a Concentration (nM) NiV-M_122B-F 500 NiV-M_122B-R 500 NiV-M_122B-FAM 100 HeV-M_85B-F 500 HeV-M_85B-R 500 HeV-M_85B-JOE 100 IC-F 50 IC-R 50 IC-Cy5 50

^a F: forward primer

R: reverse primer

IC: internal control

Production process of PCR purification kit used for PCR reaction Component Volume (μl) Primer/probe mix One 25x RT-PCR Enzyme Mix 0.8 2x RT-PCR Buffer 10 Nuclease-free Water 3.2 Template 5 Total 20

<2-3> Establishment of conditions for fast real-time PCR

In order to be utilized as a more efficient test system for detecting Nipah and Hendra viruses, it is desirable to minimize the reaction time. While the reaction time of the existing real-time polymerase chain reaction is 2 hours, the high-speed real-time polymerase chain reaction minimizes the reverse transcription process and the annealing/extension time under the existing reaction conditions to reduce the reaction time. can be shortened to 80 minutes (Table 8).

Standard real-time PCR and high-speed real-time PCR conditions cycle Temperature (℃) Standard real-time PCR Fast real-time PCR reverse transcription One 50 30 minutes 10 minutes pre-heating One 95 10 minutes 10 minutes Denaturation 40 95 15 seconds 15 seconds annealing/extension 60 1 min 30 seconds Total Time - - 120 minutes 80 minutes

As a result, it was confirmed that there was no significant difference in the experimental results (sensitivity, specificity) of the existing real-time polymerase chain reaction system and the high-speed real-time polymerase chain reaction system (Figs. 5, 6, 9 and 10), As a result, a high-speed real-time polymerase chain reaction system can be utilized for the detection of Nipa and Hendra viruses.

Nipah virus fast real-time PCR detection result during Nipah/Hendra virus multiplex real-time PCR concentration of template
(copies/μl) Standard
(Ct value) high speed
(Ct value) 1.0×10 ⁶ 18.9 19.1 1.0×10 ⁵ 22.1 22.4 1.0×10 ⁴ 25.4 25.7 1.0×10 ³ 29 29.1 1.0×10 ² 32.1 32.5 1.0×10 ¹ 34.9 35.3 1.0×10 ⁰ 38.5 (3/6) 37.5 (4/6)

Hendra virus high-speed real-time PCR detection result during Nipa/Hendra virus multiplex real-time PCR concentration of template
(copies/μl) Standard
(Ct value) high speed
(Ct value) 1.0×10 ⁶ 18.8 18.9 1.0×10 ⁵ 22 22.2 1.0×10 ⁴ 25.5 25.6 1.0×10 ³ 28.8 29 1.0×10 ² 32.1 32.3 1.0×10 ¹ 35.3 35.3 1.0×10 ⁰ 37.7 (2/6) 39.7 (1/6)

<Example 3> Confirmation of analytical sensitivity of multiple real-time polymerase chain reaction using the primer and probe set of the present invention for the purpose of detecting Nipa and Hendra viruses

Using the primer and probe set prepared in Example 1, a multiplex real-time polymerase chain reaction was performed to determine how sensitively Nipa and Hendra viruses could be detected.

Specifically, each RNA sample of Nipah and Hendra virus was obtained through in vitro transcription after oligo synthesis of the entire target amplicon as a template. Using the RNA samples, the polymerase chain reaction was performed using a real-time PCR device (Applied Biosystems 7500 Real-Time PCR System (ThermoFisher)) according to the primer and probe concentrations shown in Table 6 and the reaction conditions shown in Table 5. and was analyzed using detection software (7500 Software v2.3) (Fig. 7, Fig. 8 and Table 11).

By calculating the concentration of each synthetic DNA purified in Example 1, 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , 10 ¹ or 10 ⁰ copies/μl concentration was prepared and the polymerase chain reaction was performed. Except that, the polymerase chain reaction was performed in the same manner as described in Example 2. The minimum detection limit was evaluated based on the minimum concentration at which a band was observed with the naked eye.

Detection limit analysis of Nipa and Hendra virus detection system using multiple real-time polymerase chain reaction technology concentration of template
(copies/μl) nipa virus
(Ct value) hendra virus
(Ct value) 1.0×10 ⁶ 18.9 18.8 1.0×10 ⁵ 22.1 22.0 1.0×10 ⁴ 25.4 25.5 1.0×10 ³ 29.0 28.8 1.0×10 ² 32.1 32.1 1.0×10 ¹ 34.9 35.3 1.0×10 ⁰ 38.5 (3/6) 37.7 (2/6)

As a result, as shown in Figures 7, 8 and Table 11, the primer and probe set of the present invention can be detected even when ^{each of the RNAs of the Nipa and Hendra virus M genes are diluted to 1.0 × 10 2 copies / μl.} there was. Therefore, it can be seen that the primer and probe set of the present invention has excellent analytical sensitivity.

<Example 4> Confirmation of analytical specificity of multiple real-time polymerase chain reaction using the primer and probe set of the present invention for the purpose of detecting Nipa and Hendra viruses

In order to confirm the superiority of the analytical specificity of the real-time polymerase chain reaction using the primer set of the present invention, the analytical specificity of the multiple real-time polymerase chain reaction using the primer and probe set of the present invention was measured using RNA samples of Nipa and Hendra viruses, It was confirmed by detection of 28 kinds of pathogenic bacteria and 10 kinds of gDNA (genomic DNA) samples (Table 12) of animals including humans. RNA samples of Nipah and Hendra viruses were obtained through in vitro transcription as a template after oligo synthesis of the entire target amplicon. Pathogenic bacteria were purchased through ATCC or KCTC, and the gDNA of 10 animals was extracted after securing the animal's meat (in the case of human, Human Genomic DNA (G1521, Promega) was purchased and used).

Specifically, a multiplex real-time polymerase chain reaction was performed in the same manner as described in Example 3, except that the sample shown in Table 12 was used as a template.

Analytical Specificity of Nipah and Hendra Virus Detection System Using Multiple Real-Time Polymerase Chain Reaction Technology number name reference nepah
virus Hendra
virus One nipah virus in vitro transcribed RNA + - 2 Hendra virus in vitro transcribed RNA - + 3 Campylobacter jejuni subsp.jejuni ATCC 33560 - - 4 Campylobacter coli ATCC 33559 - - 5 Salmonella typhimurium ATCC 29629 - - 6 Salmonella typhi NCCP 12241 - - 7 Salmonella enterica subsp.enterica ATCC 14028 - - 8 Shigella flexneri KCTC 2517 - - 9 Shigella boydii KCTC 22528 - - 10 Shigella sonnei KCTC 22530 - - 11 Vibrio vunificus KCTC 2982 - - 12 Vibrio parahaemolyticus ATCC 17802 - - 13 Yersinia enterocolitica KCCM 41657 - - 14 E. coli O157 NCCP 11090 - - 15 Staphylococcus aureus ATCC 10832 - - 16 Listeria monocytogenes ATCC 19113 - - 17 Listeria innocua KCTC 3586 - - 18 Bacillus cereus ATCC 21366 - - 19 Bacillus subtilis KCTC 2213 - - 20 Bacillus thuringiensis KCTC1108 - - 21 Clostridium perfringens ATCC 12916 - - 22 Pseudomonas aeruginosa ATCC 10145 - - 23 Legionella pneumophila subsp. pneumophila KCTC 12009 - - 24 Porcine genomic DNA - - 25 Bovine genomic DNA - - 26 Sheep genomic DNA - - 27 Horse genomic DNA - - 28 Goat genomic DNA - - 29 Chicken genomic DNA - - 30 Duck genomic DNA - - 31 Donkey genomic DNA - - 32 Thurkey genomic DNA - - 33 Human genomic DNA - - 34 Dengue 1 virus MBC055 - - 35 Dengue 2 virus MBC056 - - 36 Dengue 3 virus MBC057 - - 37 Dengue 4 virus MBC058 - - 38 Chikungunya virus MBC099 - -

As a result, both Nipa and Hendra viruses of Table 12 were detected by the primer and probe set of the present invention, but no amplification reaction was detected in the pathogenic bacteria and healthy animal samples of Table 12. This suggests that the primer and probe set of the present invention can specifically detect only Nipa and Hendra viruses. In addition, the primer and probe set of the present invention can be used to simultaneously detect Nipa and Hendra viruses.

<110> KOGENEBIOTECH <120> PRIMERS AND PROBES FOR SIMULTANEOUS DETECTION OF NIPAH AND HENDRA VIRUSES AND DETECTING METHOD FOR NIPAH AND HENDRA VIRUSES USING THE SAME <130> 2020P-02-027 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nipah-M-F <400> 1 aggacaattg ctgcctaccc t 21 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Nipah-M-R <400> 2 attttctcag ttgatccagc tgttct 26 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Nipah-M-Probe <400> 3 actttgaggg aacagagttc ctccagaaga tc 32 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Hendra-M-F <400> 4 cattgccgcg taccctctt 19 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Hendra-M-R <400> 5 tgtgactttc aaagaacata gctcttc 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Hendra-M-Probe <400> 6 ttggcaagag cacttctcac cctcagg 27

Claims (17)

A primer set capable of simultaneously detecting Nipa and Hendra viruses including all the nucleotide sequences shown in SEQ ID NOs: 1, 2, 4 and 5, respectively, and a probe set including the nucleotide sequences shown in SEQ ID NOs: 3 and 6, respectively A composition for detection capable of simultaneously detecting Nipah and Hendra viruses.
The composition for detection of claim 1, wherein the primer set targets the M gene region of Nipa and Hendra viruses.
delete The composition for detection of claim 1, wherein the probe set targets the M gene region of Nipa and Hendra viruses.
[Claim 2] The composition for detection of Nipa and Hendra viruses at the same time according to claim 1, wherein the probe is further conjugated to a reporter at its 5' end.
According to claim 1, wherein the probe is further conjugated to a quencher (quencher) at its 3' end, the composition for detection capable of simultaneously detecting Nipa and Hendra viruses.
According to claim 5, wherein the reporter is FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein (fluorescein), HEX (2',4',5',7'-tetrachloro-6-carboxy- 4,7-dichlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC (fluorescein isothiocyanate), Oregon Green (oregon green), alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-X-Rhodamine), TET (Tetrachloro) -Fluorescein), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), cyanine-based dyes and thiadicarbocyanine from the group consisting of A composition for detection capable of simultaneously detecting any one or more selected Nipah and Hendra viruses.
According to claim 6, wherein the quencher is TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dapsyl ( dabcyl), Eclipse, DDQ (Deep Dark Quencher), Blackberry Quencher (Blackberry Quencher), Iowa black (Iowa black) at least one selected from the group consisting of, Nipah and Hendra virus detection composition capable of simultaneously detecting.
delete According to claim 1, wherein the detection limit of the composition is 10 ⁰ to 10 ² copies / ㎕ in the detecting composition, characterized in that, to detect a virus hendeura Nipa and at the same time.
According to claim 1, wherein the composition comprises a reverse transcriptase polymerase, a DNA polymerase, a cofactor, dATP, dCTP, dGTP and dTTP, the composition for detection capable of simultaneously detecting Nipa and Hendra virus.
A detection kit capable of simultaneously detecting Nipa and Hendra viruses comprising the composition of claim 1.
13. The method of claim 12, the detection limit of the kit 10 ⁰ to 10 ² copies / ㎕ in that, characterized in, and a detection kit for Nipa capable of detecting hendeura virus at the same time.
13. The method of claim 12, wherein the kit comprises a tube, reaction buffer, reverse transcription polymerase, DNA polymerase, dATP, dCTP, dGTP, dTTP, cofactor, DNase, RNase inhibitor, DEPC-water and sterile water. A detection kit capable of simultaneously detecting Nipah and Hendra viruses.
A method capable of simultaneously detecting Nipah and Hendra viruses comprising performing a real-time polymerase chain reaction using the primer set of claim 1 and the probe set of claim 1 as a template.
The method of claim 15 , wherein the real-time polymerase chain reaction is a high-speed real-time polymerase chain reaction.
The method according to claim 15, wherein the sample is obtained from a clinical sample or an environmental sample, and can simultaneously detect Nipah and Hendra viruses.

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