WO2021162183A1 - Lamp composition for detecting 2019 novel coronavirus, and use thereof - Google Patents

Lamp composition for detecting 2019 novel coronavirus, and use thereof Download PDF

Info

Publication number
WO2021162183A1
WO2021162183A1 PCT/KR2020/009285 KR2020009285W WO2021162183A1 WO 2021162183 A1 WO2021162183 A1 WO 2021162183A1 KR 2020009285 W KR2020009285 W KR 2020009285W WO 2021162183 A1 WO2021162183 A1 WO 2021162183A1
Authority
WO
WIPO (PCT)
Prior art keywords
novel coronavirus
detecting
isothermal amplification
lamp
mediated isothermal
Prior art date
Application number
PCT/KR2020/009285
Other languages
French (fr)
Korean (ko)
Inventor
노경태
이원식
Original Assignee
대한민국(국군의무사령부 사령관)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 대한민국(국군의무사령부 사령관) filed Critical 대한민국(국군의무사령부 사령관)
Publication of WO2021162183A1 publication Critical patent/WO2021162183A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/101—Temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/119—Reactions demanding special reaction conditions pH

Definitions

  • the present invention relates to a LAMP (Loop-mediated isothermal amplification) composition for detecting 2019 novel coronavirus and its use.
  • LAMP Loop-mediated isothermal amplification
  • the 2019 novel Coronavirus (2019-nCoV) is the causative virus of the novel coronavirus infection (Wuhan pneumonia) that occurred in December 2019, and is one of seven coronaviruses that infect humans. It has been reported that the novel coronavirus is transmitted when droplets (saliva) of an infected person penetrate the respiratory tract or the mucous membranes of the eyes, nose, and mouth. Respiratory symptoms such as cough or shortness of breath and pneumonia are the main symptoms, but asymptomatic infections are rare.
  • the World Health Organization announced on January 24, 2020 that the transmission power of the novel coronavirus is lower than that of SARS (severe acute respiratory syndrome), but higher than that of MERS (Middle East Respiratory Syndrome).
  • the gene sequence of the novel coronavirus showed the highest homology (89.1%) with the bat-derived coronavirus, 50% with MERS and 77.5% with SARS.
  • Coronaviruses are classified into four genera, including Alpha, Beta, Gamma, and Delta, and the new coronavirus was classified as belonging to the Beta group.
  • the confirmation of the new corona virus is made through the agreement between the plate coronavirus test method (Conventional PCR) and the sequencing analysis. This is to check whether the suspected patient is of the coronavirus family (pan-corona test method), and if a positive result is obtained, the virus gene sequence from the patient sample is analyzed and the test is carried out, which takes about 1 to 2 days.
  • a new test method that targets only the new coronavirus so-called 'Real Time PCR', has been applied at the Korea Centers for Disease Control and Prevention (including Incheon International Airport Quarantine Service) and 18 health and environment research institutes nationwide.
  • the core of this test method is a 'reagent kit' that can specifically diagnose the new coronavirus, not the entire coronavirus family, like the pan-corona test, and the results can be checked within 6 hours.
  • Korea Patent No. 2018079 discloses 'a primer set for detecting MERS coronavirus and its use'
  • Korean Patent No. 1100436 discloses 'a primer for detecting SARS coronavirus, a method for detecting SARS coronavirus using the same
  • the 'kit' is disclosed, there is no description about the 'LAMP composition for detecting 2019 novel coronavirus and its use' of the present invention.
  • the present invention was derived from the above needs, and the present inventors prepared a primer set for isothermal amplification specific for the N gene encoding the nucleocapsid protein of the 2019 novel coronavirus, and then the novel coronavirus infection (Wuhan pneumonia) As a result of performing Reverse Transcriptional Loop-mediated isothermal amplification (RT-LAMP) on samples of By confirming that there is, the present invention was completed.
  • R-LAMP Reverse Transcriptional Loop-mediated isothermal amplification
  • the present invention is a reverse transcriptional loop-mediated isothermal amplification for detecting the 2019 novel coronavirus (COVID-19) comprising the oligonucleotide primers of SEQ ID NOs: 1 to 6
  • a primer set for isothermal amplification is provided.
  • the present invention provides a composition for detecting 2019 novel coronavirus comprising the primer set for the reverse cycle mediated isothermal amplification.
  • the present invention provides a kit for detecting 2019 novel coronavirus, which includes a primer set for reverse cycle-mediated isothermal amplification and a reagent for performing a ring-mediated isothermal amplification reaction.
  • the present invention comprises the steps of isolating total RNA from a biological sample isolated from a patient suspected of having a 2019 novel coronavirus infection; Amplifying the target sequence using the isolated total RNA as a template and a reverse transcriptional loop-mediated isothermal amplification reaction using the primer set for reverse cycle-mediated isothermal amplification of the present invention; and detecting the product of the amplification step; provides a method of detecting 2019 novel Coronavirus, including.
  • the present invention includes the steps of pre-treating a biological sample isolated from a 2019 novel coronavirus infection suspected patient with a lysis buffer; amplifying the target sequence by a reverse cycle-mediated isothermal amplification reaction using the primer set for reverse cycle-mediated isothermal amplification of the sample pretreated with the lysis buffer; and detecting the product of the amplification step; provides a method of detecting 2019 novel Coronavirus, including.
  • the LAMP composition of the present invention and the 2019 novel coronavirus detection method using the same can check the molecular diagnostic test results within 1 hour on the spot with only a nasal swab solution and a simple real-time isothermal amplification device without gene extraction. Since the PCR test method takes about 6 hours, it can provide test results quickly, so it will be useful for the diagnosis of 2019 novel coronavirus infection.
  • FIG. 1 shows the position of the nucleotide sequence site and the RT-LAMP primer used in the preparation of the RT-LAMP primer specific for the nucleocapsid phosphoprotein coding gene of the Wuhan pneumonia virus used in the present invention.
  • FIG. 2 shows the results of RT-LAMP performed using the primer set for RT-LAMP of the present invention with the BioRad CFX 96 Real-time PCR device, showing the results of specifically amplifying only 2019-nCoV clinical samples in 22 cycles.
  • FIG. 3 shows the results of RT-LAMP performed using the primer set for RT-LAMP of the present invention with a portable real-time isothermal amplifier, and shows the results of specifically amplifying only 2019-nCoV clinical samples after 16 minutes.
  • FIG. 4 is a portable real-time isothermal amplifier as a result of Direct RT-LAMP performed using the primer set for RT-LAMP of the present invention, and the clinical specimen (sample) obtained by the nasal swab method was viewed without a separate nucleic acid extraction step. This is the result of performing isothermal amplification using the dissolution buffer developed by the inventor. After 42 minutes, only the 2019-nCoV clinical sample shows the result of specific amplification.
  • N gene is a result of homology analysis between coronaviruses of a gene encoding a nucleocapsid protein ( N gene).
  • the present invention is a reverse transcriptional loop-mediated isothermal amplification for detecting the 2019 novel coronavirus comprising the oligonucleotide primers of SEQ ID NOs: 1 to 6 , provides a primer set for RT-LAMP).
  • the term '2019 novel coronavirus' or '2019-nCoV' is the causative agent of coronavirus infection-19 (COVID-19), SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) ), and are used interchangeably.
  • the primer set for RT-LAMP for detecting 2019 novel coronavirus according to the present invention is GenBank accession no. MN908947.3, the Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, which is specific for the gene (SEQ ID NO: 7) encoding the nucleocapsid protein in the complete genome, includes an oligonucleotide primer set of SEQ ID NOs: 1 to 6.
  • the primer set according to the present invention extracts and purifies a nucleic acid from a sample (specimen), and mixes a reverse transcriptase and a polymerase using the isolated nucleic acid, particularly, RNA as a template, in one tube so that the reverse transcription reaction and the LAMP reaction occur simultaneously. It can also be used for the reverse thought-mediated isothermal amplification (RT-LAMP) method, or it can be used for a method of performing the LAMP method secondarily using cDNA made by performing reverse transcription on RNA first.
  • RT-LAMP reverse thought-mediated isothermal amplification
  • the primer set of the present invention contains 0.1 to 1.5% (v/v) of Triton X-100 and after mixing the sample with the lysis buffer, characterized in that it consists of 30 to 500 mM Tris at a pH of 6.0 to 7.0 It can also be used for the Direct RT-LAMP reaction method that directly performs LAMP without a separate nucleic acid extraction and purification process, but is not limited thereto.
  • LAMP 'Loop-mediated isothermal amplification
  • PCR polymerase chain reaction
  • the four basic primers are composed of two types of outer primers and two types of inner primers, and the outer primers are a forward outer (F3) primer and two types of backward outer (B3) primers. It is composed of and serves to unwind the DNA double strand during the non-cyclic step of the reaction.
  • the inner primer consists of two types: a forward inner primer (FIP) and a backward inner primer (BIP). It consists of the corresponding nucleotides.
  • the additional two types of primers consist of two types of forward loop (LoopF) primer and backward loop (LoopB) primer.
  • the inner primer attaches to a nucleotide sequence that does not bind to perform a loop-mediated isothermal amplification reaction. accelerate
  • the primer of SEQ ID NO: 1 is a forward outer primer F3
  • the primer of SEQ ID NO: 2 is a reverse outer primer B3
  • the primer of SEQ ID NO: 3 is a forward inner primer FIP
  • the primer of SEQ ID NO: 4 is a reverse inner primer BIP
  • the primer of SEQ ID NO: 5 is a forward loop primer LoopF
  • the primer of SEQ ID NO: 6 is a reverse loop primer LoopB.
  • the primer of the present invention may include an oligonucleotide consisting of a fragment of 15 or more, 16 or more, 17 or more consecutive nucleotides in SEQ ID NOs: 1, 2, 5 and 6, depending on the sequence length of each primer, and oligonucleotides consisting of segments of at least 32, at least 33, at least 34, at least 35, at least 36, at least 37 contiguous nucleotides in SEQ ID NOs: 3 and 4.
  • the primer (18 oligonucleotides) of SEQ ID NO: 1 may include an oligonucleotide consisting of a fragment of 15 or more, 16 or more, 17 or more nucleotides in the sequence of SEQ ID NO: 1.
  • primer refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be replicated, and may serve as a starting point for synthesis of a primer extension product.
  • the length and sequence of the primers should allow synthesis of the extension product to begin.
  • the specific length and sequence of the primer will depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.
  • the oligonucleotide used as a primer may also contain a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid or An intercalating agent may be included.
  • a nucleotide analogue for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid or An intercalating agent may be included.
  • each primer of the present invention may be modeled as a detection label in order to increase the convenience of detection.
  • the detection label may be, but is not limited to, a compound, biomolecule or biomolecule analog, etc., capable of confirming the density, concentration, amount, etc. of an amplification product in a conventional manner by linking, binding, or attaching to a primer, but is not limited thereto, but FAM, VIC , TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, TYE563, BIOTIN, DIGOXIGENIN and NED can be used.
  • the present invention also provides a composition for detecting the 2019 novel coronavirus, including a primer set for reverse cycle mediated isothermal amplification for detecting the 2019 novel coronavirus.
  • the composition for detecting 2019 novel coronavirus according to the present invention is GenBank accession no. MN908947.3, the Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, which is specific for the gene (SEQ ID NO: 7) encoding the nucleocapsid protein in the complete genome, includes an oligonucleotide primer set of SEQ ID NOs: 1 to 6.
  • the present invention also provides a kit for detecting the 2019 novel coronavirus, which includes a primer set for reverse cycle-mediated isothermal amplification for detecting the 2019 novel coronavirus and a reagent for performing a ring-mediated isothermal amplification reaction do.
  • the 2019 novel coronavirus detection kit according to the present invention is GenBank accession no. MN908947.3, the Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, which is specific for the gene (SEQ ID NO: 7) encoding the nucleocapsid protein in the complete genome, includes an oligonucleotide primer set of SEQ ID NOs: 1 to 6.
  • the 2019 novel coronavirus detection kit according to the present invention may further include a fluorescently-labeled probe capable of binding to the product amplified by the primer set for ring-mediated isothermal amplification, but is not limited thereto.
  • the reagent for performing the ring-mediated isothermal amplification reaction may include a DNA polymerase, dNTPs, and a buffer. Since the 2019 novel coronavirus is an RNA virus, a reverse cycle-mediated isothermal amplification method with an added reverse transcription process should be used for amplification. Accordingly, the kit of the present invention may further include a reverse transcriptase.
  • DNA polymerase according to an embodiment of the present invention may be a Bst polymerase, but is not limited thereto, and is mainly used in the existing loop-mediated isothermal amplification (LAMP) for reverse cycle-mediated isothermal amplification (RT-LAMP).
  • LAMP loop-mediated isothermal amplification
  • R-LAMP reverse cycle-mediated isothermal amplification
  • GspSSD DNA polymerase having a faster amplification ability and strong reverse transcriptase ability may be used instead of the Bst polymerase, but is not limited thereto.
  • the 2019 novel coronavirus detection kit of the present invention may preferably be a kit for reverse cycle mediated isothermal amplification (RT-LAMP) or a kit for direct RT-LAMP, but is not limited thereto.
  • the kit for Direct LAMP can perform the LAMP reaction immediately after mixing the sample (sample) with the lysis buffer included in the kit. There are possible features.
  • the kit of the present invention when the kit of the present invention is for Direct RT-LAMP, the kit further includes a lysis buffer, wherein the lysis buffer preferably contains 0.1 to 1.5% (v/v) of Triton X-100, pH 6.0 It may be 30-500 mM Tris of ⁇ 7.0, more preferably 300-500 mM Tris of pH 6.5, containing 0.8-1.2% (v/v) of Triton X-100, even more preferably may be 400 mM Tris, pH 6.5, containing 1% (v/v) Triton X-100, but is not limited thereto.
  • the lysis buffer preferably contains 0.1 to 1.5% (v/v) of Triton X-100, pH 6.0 It may be 30-500 mM Tris of ⁇ 7.0, more preferably 300-500 mM Tris of pH 6.5, containing 0.8-1.2% (v/v) of Triton X-100, even more preferably may be 400 mM Tris, pH
  • kit of the present invention may further include a user's guide describing optimal conditions for performing the reaction.
  • a handbook is a printout that explains how to use the kit, eg, how to prepare buffers, and suggested reaction conditions.
  • Instructions include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit.
  • the guide includes information published or provided through electronic media such as the Internet.
  • the present invention also provides a method for detecting the 2019 novel coronavirus using a primer set for reverse cycle mediated isothermal amplification for detecting the 2019 novel coronavirus including the oligonucleotide primers of SEQ ID NOs: 1 to 6.
  • the detection method according to the present invention may be performed through the RT-LAMP method or the Direct RT-LAMP method, but is not limited thereto.
  • RNA isolating total RNA from a biological sample isolated from a patient suspected of having 2019 novel coronavirus infection
  • Detecting the product of the amplification step may include, but is not limited thereto.
  • the method of the present invention includes isolating total RNA from a biological sample isolated from a patient suspected of having 2019 novel coronavirus infection.
  • the sample may be, for example, a collected human sample (nasal swab), but is not limited thereto, and is not limited if it is a biological sample isolated from a patient suspected of having 2019 novel coronavirus infection.
  • a method for isolating total RNA from the sample may use any method known in the art, for example, a phenol extraction method may be used.
  • Another detection method of 2019 novel coronavirus according to an embodiment of the present invention is,
  • Detecting the product of the amplification step may include, but is not limited thereto.
  • the step of pretreating the sample (eg, nasal swab sample or isolated human blood) with the lysis buffer according to the detection method of the present invention is preferably performed within 5 minutes after mixing the sample and the lysis buffer in a volume ratio of 1:9.
  • the lysis buffer may be 30-500 mM Tris of pH 6.0-7.0, preferably containing 0.1-1.5% (v/v) of Triton X-100, more preferably 0.8-1.2% (v/v) It may be 300-500 mM Tris at pH 6.5, including Triton X-100 of v), and even more preferably 400 mM Tris at pH 6.5, containing 1% (v/v) Triton X-100. may be, but is not limited thereto.
  • the isothermal amplification reaction may be performed at 62 ° C. to 68 ° C. for 20 minutes to 50 minutes, and preferably at 65 ° C. for 20 minutes to 50 minutes. However, it is not limited thereto.
  • the 2019 novel coronavirus detection method of the present invention includes detecting the product of the reverse thought-mediated isothermal amplification step.
  • the detection of the amplification product is fluorescence measurement using SYBR Green I, color change of an indicator such as phenol red, turbidity, precipitate formation, DNA laddering, capillary electrophoresis, DNA chip, gel electrophoresis, radiometric measurement, or phosphorescence measurement. It may be performed through, preferably, fluorescence measurement using SYBR Green I, turbidity, and DNA laddering, but is not limited thereto.
  • the primer to be used for LAMP was designed using OptiGene's LAMP Design software, and the finally designed LAMP primer for 2019 novel coronavirus detection is nucleotide sequence 401 to 700 in the nucleocapsid protein coding gene sequence consisting of the nucleotide sequence of SEQ ID NO: 7 The site is targeted, and the position of each LAMP primer sequence is as shown in FIG. 1 .
  • nucleocapsid protein-coding gene sequence consisting of the nucleotide sequence of SEQ ID NO: 7, the 401 to 700 nucleotide sequence region was converted to various 2019 coronavirus nucleotide sequences (GenBank accession no: LC521925, LR757995, LR757996, LR757997, LR757998, MN938384, MN975262, As a result of alignment with MN988668, MN988669, MN985325, MN988713, MN994467, MN994468, MN997409 and MT007544), it was confirmed that the positions targeted by the LAMP primer sets of SEQ ID NOs: 1 to 6 according to the present invention are conserved regions.
  • this F3 (SEQ ID NO: 1), B3 (SEQ ID NO: 2) and BIP primer (SEQ ID NO: 3) of the invention are different from the target nucleotide sequence and information (FIG. 5) was not expected
  • RT-LAMP using a clinical sample was performed.
  • the 2019 novel coronavirus infection clinical specimen was provided and used by the Armed Forces Capital Hospital, and to confirm whether the LMAP primer set of the present invention specifically binds to the 2019 novel coronavirus, similar clinical symptoms (cough and/or fever) were induced.
  • Adenovirus type 55, Haemophilus Influenzae , Streptococcus pneumoniae , Mycoplasma pneumoniae , Severe fever with thrombocytopenia syndrome virus, SFTS virus), malaria, and Scrub typhus infection clinical specimens (respiratory specimens) were provided by the Army Training Center and used as a comparison group.
  • RT-LAMP Reverse Transcriptional Loop-mediated Isothermal Amplification reaction consisted of 2 ⁇ l of template RNA, 1 ⁇ l of Bst polymerase (8U), 2.5 ⁇ l of 10 ⁇ buffer, 1.5 ⁇ l of 25mM dNTP, 1 ⁇ l of 10mM MgSO 4 , 5 ⁇ l of 5M betaine.
  • an isothermal amplification reaction was performed at 65° C. for 20 minutes using a BioRad CFX 96 Real time PCR device or a portable Isothermal Fluorescence PCR device, Gene-8C (ALLSHENG). Fluorescence signals were measured every 30 seconds.
  • RT-LAMP performed on a real-time PCR device
  • amplification product was confirmed only in the 2019 novel coronavirus-infected sample after 16 minutes of reaction time, and H. Influenzae , Streptococcus, until 20 minutes when the reaction was terminated.
  • Pneumoniae S. pneumoniae
  • Mycoplasma pneumoniae M. pneumonia
  • the present inventor synthesized the N gene sequences of 2019 novel coronavirus (2019-nCoV), SARS-CoV (Severe acute respiratory syndrome coronavirus) and MERS-CoV (Middle East respiratory syndrome coronavirus), respectively, and then used the sequence as a template The specificity of the primer set of the present invention was verified.
  • 2019-nCoV 2019 novel coronavirus
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • the N gene sequences of SARS-CoV and MERS-CoV used as templates are, respectively, NCBI accession no. 28,719-28,961 of the sequence MK062184.1 and NCBI accession no.
  • the 28,982 ⁇ 29,232th sequence of the MN723544.1 sequence it was prepared by requesting synthesis from Bionics Co., Ltd., and the N gene template of 2019-nCoV was prepared in the same manner as the sequence disclosed in FIG. 1 .
  • the primer sets of the present invention as shown in 6 has been confirmed that the fluorescent signal amplification from 22 cycles time for a sample containing the N gene of the 2019 swine coronavirus, the N gene of SARS-CoV and MERS-CoV In the containing sample, it was observed that the amplification reaction did not occur because the fluorescence signal was not confirmed until the reaction was terminated.
  • the primer set for RT-LAMP including the oligonucleotide primers of SEQ ID NOs: 1 to 6 prepared in the present invention can specifically detect only the 2019 novel coronavirus.
  • the detection limit of the primer sets (limit of detection, LOD) to confirm the results
  • the detection limit of LAMP primer set of the present invention is identified as 10 0 copy i.e., 1 copy sensitivity in accordance with the present invention provides real-time LAMP reaction It was found that it was very good (FIG. 7).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Virology (AREA)

Abstract

The present invention relates to a loop-mediated isothermal amplification (LAMP) composition for detecting the 2019 novel coronavirus, and a use thereof. A 2019 novel coronavirus detection method using the LAMP composition of the present invention can confirm a molecular diagnosis test result within an hour on-site by using a simple real-time isothermal amplification device only without separate nucleic acid extraction and purification processes from a sample, and thus can be effectively used in point-of-care testing and the like.

Description

2019 신종코로나바이러스 검출용 LAMP 조성물 및 이의 용도LAMP composition for 2019 novel coronavirus detection and use thereof
본 발명은 2019 신종코로나바이러스 검출용 LAMP(Loop-mediated isothermal amplification) 조성물 및 이의 용도에 관한 것이다.The present invention relates to a LAMP (Loop-mediated isothermal amplification) composition for detecting 2019 novel coronavirus and its use.
신종코로나바이러스(2019 novel Coronavirus, 2019-nCoV)는 2019년 12월 발생한 신종코로나바이러스감염증(우한 폐렴)의 원인 바이러스로, 인체 감염 7개 코로나바이러스 중 하나다. 신종코로나바이러스는 감염자의 비말(침방울)이 호흡기나 눈·코·입의 점막으로 침투될 때 전염되는 것으로 보고되었으며, 감염되면 약 2~14일(추정)의 잠복기를 거친 뒤 발열(37.5도) 및 기침이나 호흡곤란 등 호흡기 증상, 폐렴이 주 증상으로 나타나지만 무증상 감염 사례도 드물게 나오고 있다. 세계보건기구(WHO)는 2020년 1월 24일 신종코로나바이러스의 전파력이 사스(SARS, 중증급성호흡기증후군)보다는 낮지만, 메르스(MERS, 중동호흡기증후군)보다는 높은 것으로 파악한다고 밝혔다.The 2019 novel Coronavirus (2019-nCoV) is the causative virus of the novel coronavirus infection (Wuhan pneumonia) that occurred in December 2019, and is one of seven coronaviruses that infect humans. It has been reported that the novel coronavirus is transmitted when droplets (saliva) of an infected person penetrate the respiratory tract or the mucous membranes of the eyes, nose, and mouth. Respiratory symptoms such as cough or shortness of breath and pneumonia are the main symptoms, but asymptomatic infections are rare. The World Health Organization (WHO) announced on January 24, 2020 that the transmission power of the novel coronavirus is lower than that of SARS (severe acute respiratory syndrome), but higher than that of MERS (Middle East Respiratory Syndrome).
질병관리본부의 보고에 따르면 신종코로나바이러스의 유전자 염기서열은 박쥐 유래 유사 코로나바이러스와 가장 높은 상동성(89.1%)을 보였으며, 메르스와는 50%, 사스와는 77.5%의 상동성이 나타났다. 코로나바이러스는 알파(Alpha)·베타(Beta)·감마(Gamma)·델타(Delta) 등 4속(屬)으로 분류되는데, 신종코로나바이러스는 베타(Beta)군에 속하는 것으로 분류되었다. 현재까지 확인된 인체 전염 코로나바이러스는 총 7종으로 HCoV 229E, HCoV NL63, HCoV OC43, HCoV HKU1, SARS-CoV, MERS-CoV, 2019-nCoV가 이에 해당한다.According to a report from the Korea Centers for Disease Control and Prevention, the gene sequence of the novel coronavirus showed the highest homology (89.1%) with the bat-derived coronavirus, 50% with MERS and 77.5% with SARS. Coronaviruses are classified into four genera, including Alpha, Beta, Gamma, and Delta, and the new coronavirus was classified as belonging to the Beta group. There are a total of seven types of coronaviruses that have been identified so far, including HCoV 229E, HCoV NL63, HCoV OC43, HCoV HKU1, SARS-CoV, MERS-CoV, and 2019-nCoV.
현재, 신종코로나 확진 여부는 판 코로나바이러스 검사법(Conventional PCR)과 염기서열분석 일치 여부를 통해 이뤄지고 있다. 이는 의심환자에 대해 코로나바이러스 계열인지 여부(판코로나 검사법)를 확인한 뒤 양성반응이 나오면 환자 검체에서 나온 바이러스 유전자 염기서열을 분석해 검사를 진행하는 것으로, 약 1~2일이 소요된다. 그러나 2020년 1월 31일부터는 신종코로나바이러스만을 타깃으로 하는 새 검사법, 이른바 'Real Time PCR'이 질병관리본부(국립인천공항검역소 포함)와 전국 18개 보건환경연구원에서 적용되었다. 이 검사법은 판 코로나 검사처럼 코로나바이러스 전체 계열이 아닌 신종코로나바이러스를 특정해 진단할 수 있는 '시약 키트'가 핵심으로, 검사 6시간 이내 결과를 확인할 수 있다.Currently, the confirmation of the new corona virus is made through the agreement between the plate coronavirus test method (Conventional PCR) and the sequencing analysis. This is to check whether the suspected patient is of the coronavirus family (pan-corona test method), and if a positive result is obtained, the virus gene sequence from the patient sample is analyzed and the test is carried out, which takes about 1 to 2 days. However, from January 31, 2020, a new test method that targets only the new coronavirus, so-called 'Real Time PCR', has been applied at the Korea Centers for Disease Control and Prevention (including Incheon International Airport Quarantine Service) and 18 health and environment research institutes nationwide. The core of this test method is a 'reagent kit' that can specifically diagnose the new coronavirus, not the entire coronavirus family, like the pan-corona test, and the results can be checked within 6 hours.
한편, 한국등록특허 제2018079호에는 '메르스 코로나바이러스 검출용 프라이머 세트 및 이의 용도'가 개시되어 있고, 한국등록특허 제1100436호에는 '사스 코로나바이러스 검출용 프라이머, 그를 이용한 사스 코로나바이러스 검출 방법 및 키트'가 개시되어 있으나, 본 발명의 '2019신종코로나바이러스 검출용 LAMP 조성물 및 이의 용도'에 대해서는 기재된 바가 없다.On the other hand, Korea Patent No. 2018079 discloses 'a primer set for detecting MERS coronavirus and its use', and Korean Patent No. 1100436 discloses 'a primer for detecting SARS coronavirus, a method for detecting SARS coronavirus using the same, and Although the 'kit' is disclosed, there is no description about the 'LAMP composition for detecting 2019 novel coronavirus and its use' of the present invention.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 2019 신종코로나바이러스의 뉴클레오캡시드 단백질을 코딩하는 N 유전자에 특이적인 등온증폭용 프라이머 세트를 제작한 후 신종코로나바이러스감염증(우한 폐렴)의 시료를 대상으로 역전사고리매개등온증폭반응(Reverse Transcriptional Loop-mediated isothermal amplification, RT-LAMP)을 수행한 결과, 시료 내에서 신종코로나바이러스(2019 novel Coronavirus)의 핵산을 비특이적 반응 없이 정확하게 검출할 수 있음을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above needs, and the present inventors prepared a primer set for isothermal amplification specific for the N gene encoding the nucleocapsid protein of the 2019 novel coronavirus, and then the novel coronavirus infection (Wuhan pneumonia) As a result of performing Reverse Transcriptional Loop-mediated isothermal amplification (RT-LAMP) on samples of By confirming that there is, the present invention was completed.
상기 과제를 해결하기 위해, 본 발명은 서열번호 1 내지 6의 올리고뉴클레오티드 프라이머를 포함하는 2019 신종코로나바이러스(2019 novel Coronavirus, COVID-19)를 검출하기 위한 역전사고리매개등온증폭(Reverse transcriptional Loop-mediated isothermal amplification)용 프라이머 세트를 제공한다.In order to solve the above problems, the present invention is a reverse transcriptional loop-mediated isothermal amplification for detecting the 2019 novel coronavirus (COVID-19) comprising the oligonucleotide primers of SEQ ID NOs: 1 to 6 A primer set for isothermal amplification is provided.
또한, 본 발명은 상기 역전사고리매개등온증폭용 프라이머 세트를 포함하는 2019 신종코로나바이러스 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting 2019 novel coronavirus comprising the primer set for the reverse cycle mediated isothermal amplification.
또한, 본 발명은 상기 역전사고리매개등온증폭용 프라이머 세트 및 고리매개등온증폭 반응을 수행하기 위한 시약을 포함하는 2019 신종코로나바이러스 검출용 키트를 제공한다.In addition, the present invention provides a kit for detecting 2019 novel coronavirus, which includes a primer set for reverse cycle-mediated isothermal amplification and a reagent for performing a ring-mediated isothermal amplification reaction.
또한, 본 발명은 2019 신종코로나바이러스감염증 의심 환자로부터 분리된 생물학적 시료로부터 총 RNA를 분리하는 단계; 상기 분리된 총 RNA를 주형으로 하고, 본 발명의 역전사고리매개등온증폭용 프라이머 세트를 이용하여 역전사고리매개등온증폭(Reverse transcriptional Loop-mediated Isothermal Amplification) 반응으로 표적서열을 증폭하는 단계; 및 상기 증폭 단계의 산물을 검출하는 단계;를 포함하는 2019 신종코로나바이러스(2019 novel Coronavirus)를 검출하는 방법을 제공한다.In addition, the present invention comprises the steps of isolating total RNA from a biological sample isolated from a patient suspected of having a 2019 novel coronavirus infection; Amplifying the target sequence using the isolated total RNA as a template and a reverse transcriptional loop-mediated isothermal amplification reaction using the primer set for reverse cycle-mediated isothermal amplification of the present invention; and detecting the product of the amplification step; provides a method of detecting 2019 novel Coronavirus, including.
또한, 본 발명은 2019 신종코로나바이러스감염증 의심 환자로부터 분리된 생물학적 시료를 용해 버퍼로 전처리하는 단계; 상기 용해 버퍼로 전처리된 시료를 본 발명의 역전사고리매개등온증폭용 프라이머 세트를 이용하여 역전사고리매개등온증폭 반응으로 표적서열을 증폭하는 단계; 및 상기 증폭 단계의 산물을 검출하는 단계;를 포함하는 2019 신종코로나바이러스(2019 novel Coronavirus)를 검출하는 방법을 제공한다.In addition, the present invention includes the steps of pre-treating a biological sample isolated from a 2019 novel coronavirus infection suspected patient with a lysis buffer; amplifying the target sequence by a reverse cycle-mediated isothermal amplification reaction using the primer set for reverse cycle-mediated isothermal amplification of the sample pretreated with the lysis buffer; and detecting the product of the amplification step; provides a method of detecting 2019 novel Coronavirus, including.
본 발명의 LAMP 조성물 및 이를 이용한 2019 신종코로나바이러스 검출 방법은 유전자 추출없이 코 면봉법(nasal swab) 용액과 간단한 실시간 등온증폭기기만으로 분자진단 검사 결과를 현장에서 1시간 이내에 확인할 수 있으므로, 현행 Real time PCR 검사법이 6시간 정도 소요되는데 비해 신속하게 검사 결과를 제공할 수 있으므로, 2019 신종코로나바이러스감염증의 진단에 유용하게 활용될 수 있을 것이다.The LAMP composition of the present invention and the 2019 novel coronavirus detection method using the same can check the molecular diagnostic test results within 1 hour on the spot with only a nasal swab solution and a simple real-time isothermal amplification device without gene extraction. Since the PCR test method takes about 6 hours, it can provide test results quickly, so it will be useful for the diagnosis of 2019 novel coronavirus infection.
도 1은 본 발명에서 사용한 우한폐렴 바이러스의 뉴클레오캡시드 단백질(nucleocapsid phosphoprotein) 코딩 유전자에 특이적인 RT-LAMP용 프라이머 제작에 사용된 염기서열 부위와 RT-LAMP용 프라이머의 위치를 나타낸 것이다.1 shows the position of the nucleotide sequence site and the RT-LAMP primer used in the preparation of the RT-LAMP primer specific for the nucleocapsid phosphoprotein coding gene of the Wuhan pneumonia virus used in the present invention.
도 2는 BioRad CFX 96 Real time PCR 기기로 본 발명의 RT-LAMP용 프라이머 세트를 이용하여 수행한 RT-LAMP의 결과로, 22 cycles에서 2019-nCoV 임상검체만 특이적으로 증폭되는 결과를 보여준다.FIG. 2 shows the results of RT-LAMP performed using the primer set for RT-LAMP of the present invention with the BioRad CFX 96 Real-time PCR device, showing the results of specifically amplifying only 2019-nCoV clinical samples in 22 cycles.
도 3은 휴대용 Real time 등온증폭기로 본 발명의 RT-LAMP용 프라이머 세트를 이용하여 수행한 RT-LAMP의 결과로, 16분 이후부터 2019-nCoV 임상검체만 특이적으로 증폭되는 결과를 보여준다.3 shows the results of RT-LAMP performed using the primer set for RT-LAMP of the present invention with a portable real-time isothermal amplifier, and shows the results of specifically amplifying only 2019-nCoV clinical samples after 16 minutes.
도 4는 휴대용 Real time 등온증폭기로 본 발명의 RT-LAMP용 프라이머 세트를 이용하여 수행한 Direct RT-LAMP의 결과로, 코 면봉법으로 획득한 임상검체(시료)를 별도의 핵산 추출 단계없이 본 발명자가 개발한 용해 버퍼를 이용하여 등온증폭을 수행한 결과이다. 42분 이후부터 2019-nCoV 임상검체만 특이적으로 증폭되는 결과를 보여준다.4 is a portable real-time isothermal amplifier as a result of Direct RT-LAMP performed using the primer set for RT-LAMP of the present invention, and the clinical specimen (sample) obtained by the nasal swab method was viewed without a separate nucleic acid extraction step. This is the result of performing isothermal amplification using the dissolution buffer developed by the inventor. After 42 minutes, only the 2019-nCoV clinical sample shows the result of specific amplification.
도 5는 뉴클레오캡시드 단백질을 코딩하는 유전자( N 유전자)의 코로나바이러스 간 상동성 분석 결과이다.5 is a result of homology analysis between coronaviruses of a gene encoding a nucleocapsid protein ( N gene).
도 6은 2019-nCoV, SARS-CoV 및 MERS-CoV의 N 유전자 염기서열 주형을 대상으로 본 발명에 따른 RT-LAMP용 프라이머 세트의 특이성을 확인한 결과이다.6 is a result confirming the specificity of the primer set for RT-LAMP according to the present invention for the N gene sequence template of 2019-nCoV, SARS-CoV and MERS-CoV.
도 7은 본 발명에 따른 RT-LAMP용 프라이머 세트의 검출 한계(LOD)를 확인한 결과이다. 서열번호 7의 염기서열로 이루어진 2019 신종코로나바이러스 N 유전자 서열에서 401~700번째 염기서열 부위를 합성하여 base pair 및 핵산량을 계산하여 카피 수를 환산하여 사용하였다.7 is a result of confirming the limit of detection (LOD) of the primer set for RT-LAMP according to the present invention. In the 2019 novel coronavirus N gene sequence consisting of the nucleotide sequence of SEQ ID NO: 7, the 401-700th nucleotide sequence was synthesized, the base pair and the amount of nucleic acid were calculated, and the copy number was converted and used.
본 발명의 목적을 달성하기 위하여, 본 발명은 서열번호 1 내지 6의 올리고뉴클레오티드 프라이머를 포함하는 2019 신종코로나바이러스(2019 novel Coronavirus)를 검출하기 위한 역전사고리매개등온증폭(Reverse transcriptional Loop-mediated isothermal amplification, RT-LAMP)용 프라이머 세트를 제공한다.In order to achieve the object of the present invention, the present invention is a reverse transcriptional loop-mediated isothermal amplification for detecting the 2019 novel coronavirus comprising the oligonucleotide primers of SEQ ID NOs: 1 to 6 , provides a primer set for RT-LAMP).
본 명세서에서 사용된 용어 '2019 신종코로나바이러스(2019 novel Coronavirus)' 또는 '2019-nCoV'는 코로나바이러스감염증-19 (COVID-19)의 원인 병원체인 SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2)를 의미하는 것으로, 서로 혼용되어 사용되었다.As used herein, the term '2019 novel coronavirus' or '2019-nCoV' is the causative agent of coronavirus infection-19 (COVID-19), SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) ), and are used interchangeably.
본 발명에 따른 2019 신종코로나바이러스를 검출하기 위한 RT-LAMP용 프라이머 세트는 GenBank accession no. MN908947.3인 Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, complete genome에서 뉴클레오캡시드 단백질을 코딩하는 유전자(서열번호 7)에 특이적인, 서열번호 1 내지 6의 올리고뉴클레오티드 프라이머 세트를 포함한다.The primer set for RT-LAMP for detecting 2019 novel coronavirus according to the present invention is GenBank accession no. MN908947.3, the Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, which is specific for the gene (SEQ ID NO: 7) encoding the nucleocapsid protein in the complete genome, includes an oligonucleotide primer set of SEQ ID NOs: 1 to 6.
본 발명에 따른 프라이머 세트는 시료(검체)로부터 핵산을 추출 및 정제하여, 분리된 핵산 특히, RNA를 주형으로 역전사 효소와 중합효소를 하나의 튜브에 혼합하여 역전사 반응과 LAMP 반응이 동시에 일어나도록 수행하는 역전사고리매개등온증폭(RT-LAMP) 방법에 이용될 수도 있고, RNA를 대상으로 역전사를 먼저 수행하여 만들어진 cDNA를 이용하여 2차적으로 LAMP법을 수행하는 방법에도 이용될 수 있다. 또한, 본 발명의 상기 프라이머 세트는 0.1~1.5 %(v/v)의 Triton X-100을 포함하며 pH 6.0~7.0의 30~500 mM Tris로 이루어진 것을 특징으로 하는 용해 버퍼와 시료를 혼합한 후 별도의 핵산 추출 및 정제 과정 없이 바로 LAMP를 수행하는 Direct RT-LAMP 반응법에도 이용될 수 있으나, 이에 제한되지 않는다.The primer set according to the present invention extracts and purifies a nucleic acid from a sample (specimen), and mixes a reverse transcriptase and a polymerase using the isolated nucleic acid, particularly, RNA as a template, in one tube so that the reverse transcription reaction and the LAMP reaction occur simultaneously. It can also be used for the reverse thought-mediated isothermal amplification (RT-LAMP) method, or it can be used for a method of performing the LAMP method secondarily using cDNA made by performing reverse transcription on RNA first. In addition, the primer set of the present invention contains 0.1 to 1.5% (v/v) of Triton X-100 and after mixing the sample with the lysis buffer, characterized in that it consists of 30 to 500 mM Tris at a pH of 6.0 to 7.0 It can also be used for the Direct RT-LAMP reaction method that directly performs LAMP without a separate nucleic acid extraction and purification process, but is not limited thereto.
본 명세서에 있어서, 용어 '고리매개등온증폭(Loop-mediated isothermal amplification, LAMP)'은 기존 PCR(polymerase chain reaction) 법과 달리 등온의 조건에서 증폭 반응을 수행할 수 있는 방법을 의미한다. LAMP 반응을 위해서는 기본적으로 4종의 프라이머(F3, B3, FIP, BIP)가 필요하고, 반응 속도를 향상시키기 위해 2종의 프라이머(LF, LB)를 추가하여 최종 6종의 각기 다른 염기서열로 이루어진 올리고뉴클레오티드 프라이머가 반응에 필요하다.As used herein, the term 'Loop-mediated isothermal amplification (LAMP)' refers to a method capable of performing an amplification reaction under isothermal conditions, unlike the existing polymerase chain reaction (PCR) method. For the LAMP reaction, basically 4 types of primers (F3, B3, FIP, BIP) are required, and to improve the reaction rate, 2 types of primers (LF, LB) are added to obtain the final 6 types of different nucleotide sequences. A constructed oligonucleotide primer is required for the reaction.
상기 4종의 기본 프라이머는 외부(outer) 프라이머 2종과 내부(inner) 프라이머 2종으로 구성되며, 외부 프라이머는 정방향 외부(forward outer, F3) 프라이머와 역방향 외부(backward outer, B3) 프라이머 2종으로 구성되고 반응의 비순환기(non-cyclic step) 동안 DNA 이중 가닥을 풀어주는 역할을 한다. 내부 프라이머는 정방향 내부 프라이머(forward inner primer, FIP)와 역방향 내부 프라이머(backward inner primer, BIP) 2종으로 구성되고 고리매개 등온증폭반응에 필수적인 고리(loop)를 만들 수 있도록 정방향 및 역방향 염기서열에 해당하는 뉴클레오티드로 구성된다. 추가 2종의 프라이머는 정방향 고리(forward loop, LoopF) 프라이머와 역방향 고리(backward loop, LoopB) 프라이머 2종으로 구성되며 내부(inner) 프라이머가 결합하지 않는 염기서열에 부착하여 고리매개등온증폭 반응을 가속화시킨다.The four basic primers are composed of two types of outer primers and two types of inner primers, and the outer primers are a forward outer (F3) primer and two types of backward outer (B3) primers. It is composed of and serves to unwind the DNA double strand during the non-cyclic step of the reaction. The inner primer consists of two types: a forward inner primer (FIP) and a backward inner primer (BIP). It consists of the corresponding nucleotides. The additional two types of primers consist of two types of forward loop (LoopF) primer and backward loop (LoopB) primer. The inner primer attaches to a nucleotide sequence that does not bind to perform a loop-mediated isothermal amplification reaction. accelerate
본 발명의 서열번호 1 내지 6의 올리고뉴클레오티드 프라이머로 이루어진 프라이머 세트에 있어서, 서열번호 1의 프라이머는 정방향 외부 프라이머인 F3이고, 서열번호 2의 프라이머는 역방향 외부 프라이머인 B3이고, 서열번호 3의 프라이머는 정방향 내부 프라이머인 FIP이며, 서열번호 4의 프라이머는 역방향 내부 프라이머인 BIP이며, 서열번호 5의 프라이머는 정방향 고리 프라이머인 LoopF이고, 서열번호 6의 프라이머는 역방향 고리 프라이머인 LoopB이다.In the primer set consisting of the oligonucleotide primers of SEQ ID NOs: 1 to 6 of the present invention, the primer of SEQ ID NO: 1 is a forward outer primer F3, the primer of SEQ ID NO: 2 is a reverse outer primer B3, and the primer of SEQ ID NO: 3 is a forward inner primer FIP, the primer of SEQ ID NO: 4 is a reverse inner primer BIP, the primer of SEQ ID NO: 5 is a forward loop primer LoopF, and the primer of SEQ ID NO: 6 is a reverse loop primer LoopB.
본 발명의 상기 프라이머는, 각 프라이머의 서열 길이에 따라, 서열번호 1, 2, 5 및 6 내의 15개 이상, 16개 이상, 17개 이상의 연속 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드를 포함할 수 있고, 서열번호 3 및 4 내의 32개 이상, 33개 이상, 34개 이상, 35개 이상, 36개 이상, 37개 이상의 연속 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드를 포함할 수 있다. 예를 들면, 서열번호 1의 프라이머(18개 올리고뉴클레오티드)는 서열번호 1의 서열 내의 15개 이상, 16개 이상, 17개 이상의 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드를 포함할 수 있다.The primer of the present invention may include an oligonucleotide consisting of a fragment of 15 or more, 16 or more, 17 or more consecutive nucleotides in SEQ ID NOs: 1, 2, 5 and 6, depending on the sequence length of each primer, and oligonucleotides consisting of segments of at least 32, at least 33, at least 34, at least 35, at least 36, at least 37 contiguous nucleotides in SEQ ID NOs: 3 and 4. For example, the primer (18 oligonucleotides) of SEQ ID NO: 1 may include an oligonucleotide consisting of a fragment of 15 or more, 16 or more, 17 or more nucleotides in the sequence of SEQ ID NO: 1.
본 명세서에 있어서, 용어 "프라이머"는 복제하려는 핵산 가닥에 상보적인 단일 가닥의 올리고뉴클레오티드 서열을 말하며, 프라이머 연장 산물의 합성을 위한 개시점으로서 작용할 수 있다. 상기 프라이머의 길이 및 서열은 연장 산물의 합성을 시작하도록 허용해야 한다. 프라이머의 구체적인 길이 및 서열은 요구되는 DNA 또는 RNA 표적의 복합도(complexity) 뿐만 아니라 온도 및 이온 강도와 같은 프라이머 이용 조건에 의존할 것이다.As used herein, the term “primer” refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be replicated, and may serve as a starting point for synthesis of a primer extension product. The length and sequence of the primers should allow synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.
본 발명에 있어서, 프라이머로서 이용된 올리고뉴클레오티드는 또한 뉴클레오티드 유사체(analogue), 예를 들면, 포스포로티오에이트(phosphorothioate), 알킬포스포로티오에이트 또는 펩티드 핵산(peptide nucleic acid)을 포함할 수 있거나 또는 삽입 물질(intercalating agent)을 포함할 수 있다.In the present invention, the oligonucleotide used as a primer may also contain a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid or An intercalating agent may be included.
또한, 본 발명의 각 프라이머에는 검출의 편의성을 높이기 위해, 검출용 표지로 모식될 수 있다. 검출용 표지는 프라이머에 연결, 결합, 또는 부착시켜 통상적인 방식으로 증폭산물의 밀도, 농도, 양 등을 확인할 수 있는 화합물, 생체 분자 또는 생체 분자 유사체 등일 수 있으며, 이에 한정하지 않으나, FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, TYE563, BIOTIN, DIGOXIGENIN 및 NED 등이 사용될 수 있다.In addition, each primer of the present invention may be modeled as a detection label in order to increase the convenience of detection. The detection label may be, but is not limited to, a compound, biomolecule or biomolecule analog, etc., capable of confirming the density, concentration, amount, etc. of an amplification product in a conventional manner by linking, binding, or attaching to a primer, but is not limited thereto, but FAM, VIC , TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, TYE563, BIOTIN, DIGOXIGENIN and NED can be used.
본 발명은 또한, 상기 2019 신종코로나바이러스를 검출하기 위한 역전사고리매개등온증폭용 프라이머 세트를 포함하는 2019 신종코로나바이러스(2019 novel Coronavirus) 검출용 조성물을 제공한다. 본 발명에 따른 2019 신종코로나바이러스 검출용 조성물은 GenBank accession no. MN908947.3인 Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, complete genome에서 뉴클레오캡시드 단백질을 코딩하는 유전자(서열번호 7)에 특이적인, 서열번호 1 내지 6의 올리고뉴클레오티드 프라이머 세트를 포함한다.The present invention also provides a composition for detecting the 2019 novel coronavirus, including a primer set for reverse cycle mediated isothermal amplification for detecting the 2019 novel coronavirus. The composition for detecting 2019 novel coronavirus according to the present invention is GenBank accession no. MN908947.3, the Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, which is specific for the gene (SEQ ID NO: 7) encoding the nucleocapsid protein in the complete genome, includes an oligonucleotide primer set of SEQ ID NOs: 1 to 6.
본 발명은 또한, 상기 2019 신종코로나바이러스를 검출하기 위한 역전사고리매개등온증폭용 프라이머 세트 및 고리매개등온증폭 반응을 수행하기 위한 시약을 포함하는 2019 신종코로나바이러스(2019 novel Coronavirus) 검출용 키트를 제공한다.The present invention also provides a kit for detecting the 2019 novel coronavirus, which includes a primer set for reverse cycle-mediated isothermal amplification for detecting the 2019 novel coronavirus and a reagent for performing a ring-mediated isothermal amplification reaction do.
본 발명에 따른 2019 신종코로나바이러스 검출용 키트는 GenBank accession no. MN908947.3인 Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, complete genome에서 뉴클레오캡시드 단백질을 코딩하는 유전자(서열번호 7)에 특이적인, 서열번호 1 내지 6의 올리고뉴클레오티드 프라이머 세트를 포함한다.The 2019 novel coronavirus detection kit according to the present invention is GenBank accession no. MN908947.3, the Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, which is specific for the gene (SEQ ID NO: 7) encoding the nucleocapsid protein in the complete genome, includes an oligonucleotide primer set of SEQ ID NOs: 1 to 6.
또한, 본 발명에 따른 2019 신종코로나바이러스 검출용 키트는 상기 고리매개등온증폭용 프라이머 세트에 의해 증폭된 산물에 결합할 수 있는, 형광 표지된 프로브를 추가로 포함할 수 있으나, 이에 제한되지 않는다.In addition, the 2019 novel coronavirus detection kit according to the present invention may further include a fluorescently-labeled probe capable of binding to the product amplified by the primer set for ring-mediated isothermal amplification, but is not limited thereto.
본 발명의 키트에서, 상기 고리매개등온증폭 반응을 수행하기 위한 시약은 DNA 중합효소, dNTPs 및 버퍼 등을 포함할 수 있다. 2019 신종코로나바이러스는 RNA 바이러스이기 때문에 증폭을 위해서는 역전사(reverse transcription) 과정이 추가된 역전사고리매개등온증폭법이 이용되어야 한다. 따라서 본 발명의 키트는 역전사 효소를 추가로 포함할 수 있다.In the kit of the present invention, the reagent for performing the ring-mediated isothermal amplification reaction may include a DNA polymerase, dNTPs, and a buffer. Since the 2019 novel coronavirus is an RNA virus, a reverse cycle-mediated isothermal amplification method with an added reverse transcription process should be used for amplification. Accordingly, the kit of the present invention may further include a reverse transcriptase.
본 발명의 일 구현 예에 따른 DNA 중합효소는 Bst 중합효소일 수 있으나, 이에 제한되지 않으며, 역전사고리매개등온증폭법(RT-LAMP)을 위해 기존의 고리매개등온증폭법(LAMP)에서 주로 사용되어진 Bst 중합효소 대신 보다 빠른 증폭 능력과 강력한 역전사효소능을 가지고 있는 GspSSD DNA 중합효소 등를 사용할 수 있으나, 이에 제한되지 않는다.DNA polymerase according to an embodiment of the present invention may be a Bst polymerase, but is not limited thereto, and is mainly used in the existing loop-mediated isothermal amplification (LAMP) for reverse cycle-mediated isothermal amplification (RT-LAMP). GspSSD DNA polymerase having a faster amplification ability and strong reverse transcriptase ability may be used instead of the Bst polymerase, but is not limited thereto.
본 발명의 2019 신종코로나바이러스 검출용 키트는 바람직하게는 역전사고리매개등온증폭(RT-LAMP)용 키트 또는 Direct RT-LAMP용 키트일 수 있으나, 이에 제한되지 않는다. Direct LAMP용 키트는 시료(검체)를 키트에 포함된 용해 버퍼와 혼합한 후 LAMP 반응을 바로 수행할 수 있어, 시료로부터 핵산을 추출하고 정제하는 단계가 필요한 종래의 LAMP 진단법에 비해, 신속한 진단이 가능한 특징이 있다.The 2019 novel coronavirus detection kit of the present invention may preferably be a kit for reverse cycle mediated isothermal amplification (RT-LAMP) or a kit for direct RT-LAMP, but is not limited thereto. The kit for Direct LAMP can perform the LAMP reaction immediately after mixing the sample (sample) with the lysis buffer included in the kit. There are possible features.
본 발명의 키트가 Direct RT-LAMP용일 경우, 상기 키트는 용해 버퍼를 추가로 포함하며, 상기 용해 버퍼는 바람직하게는 0.1~1.5 %(v/v)의 Triton X-100을 포함하는, pH 6.0~7.0의 30~500 mM Tris일 수 있고, 더욱 바람직하게는 0.8~1.2 %(v/v)의 Triton X-100을 포함하는, pH 6.5의 300~500 mM Tris일 수 있으며, 더 더욱 바람직하게는 1 %(v/v)의 Triton X-100을 포함하는, pH 6.5의 400 mM Tris일 수 있으나, 이에 제한되지 않는다.When the kit of the present invention is for Direct RT-LAMP, the kit further includes a lysis buffer, wherein the lysis buffer preferably contains 0.1 to 1.5% (v/v) of Triton X-100, pH 6.0 It may be 30-500 mM Tris of ˜7.0, more preferably 300-500 mM Tris of pH 6.5, containing 0.8-1.2% (v/v) of Triton X-100, even more preferably may be 400 mM Tris, pH 6.5, containing 1% (v/v) Triton X-100, but is not limited thereto.
또한, 본 발명의 키트는 최적의 반응 수행 조건을 기재한 사용자 안내서를 추가로 포함할 수 있다. 안내서는 키트 사용법, 예를 들면, 완충액 제조 방법, 제시되는 반응 조건 등을 설명하는 인쇄물이다. 안내서는 팜플렛 또는 전단지 형태의 안내 책자, 키트에 부착된 라벨, 및 키트를 포함하는 패키지의 표면상에 설명을 포함한다. 또한, 안내서는 인터넷과 같이 전기 매체를 통해 공개되거나 제공되는 정보를 포함한다.In addition, the kit of the present invention may further include a user's guide describing optimal conditions for performing the reaction. A handbook is a printout that explains how to use the kit, eg, how to prepare buffers, and suggested reaction conditions. Instructions include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information published or provided through electronic media such as the Internet.
본 발명은 또한, 상기 서열번호 1 내지 6의 올리고뉴클레오티드 프라이머를 포함하는 2019 신종코로나바이러스를 검출하기 위한 역전사고리매개등온증폭용 프라이머 세트를 이용한 2019 신종코로나바이러스를 검출하는 방법을 제공한다. 본 발명에 따른 상기 검출 방법은 RT-LAMP 방법 또는 Direct RT-LAMP 방법을 통해 수행될 수 있으나, 이에 제한되지 않는다.The present invention also provides a method for detecting the 2019 novel coronavirus using a primer set for reverse cycle mediated isothermal amplification for detecting the 2019 novel coronavirus including the oligonucleotide primers of SEQ ID NOs: 1 to 6. The detection method according to the present invention may be performed through the RT-LAMP method or the Direct RT-LAMP method, but is not limited thereto.
본 발명의 일 구현 예에 따른 2019 신종코로나바이러스 검출 방법은,The 2019 novel coronavirus detection method according to an embodiment of the present invention,
2019 신종코로나바이러스감염증 의심 환자로부터 분리된 생물학적 시료로부터 총 RNA를 분리하는 단계;isolating total RNA from a biological sample isolated from a patient suspected of having 2019 novel coronavirus infection;
상기 분리된 총 RNA를 주형으로 하고, 본 발명의 역전사고리매개등온증폭용 프라이머 세트를 이용하여 역전사고리매개등온증폭(Reverse transcriptional Loop-mediated Isothermal Amplification) 반응으로 표적서열을 증폭하는 단계; 및Amplifying the target sequence using the isolated total RNA as a template and a reverse transcriptional loop-mediated isothermal amplification reaction using the primer set for reverse cycle-mediated isothermal amplification of the present invention; and
상기 증폭 단계의 산물을 검출하는 단계;를 포함할 수 있으나, 이에 제한되지 않는다.Detecting the product of the amplification step; may include, but is not limited thereto.
본 발명의 방법은 2019 신종코로나바이러스감염증 의심 환자로부터 분리된 생물학적 시료에서 총 RNA를 분리하는 단계를 포함한다. 상기 시료는 예를 들면, 채취한 인간의 검체(코 면봉법(nasal swab))일 수 있으나, 이에 한정되지 않으며, 2019 신종코로나바이러스감염증 의심 환자로부터 분리된 생물학적 시료이면 제한되지 않는다. 상기 시료에서 총 RNA를 분리하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있으며, 예를 들면, 페놀 추출 방법을 이용할 수 있다.The method of the present invention includes isolating total RNA from a biological sample isolated from a patient suspected of having 2019 novel coronavirus infection. The sample may be, for example, a collected human sample (nasal swab), but is not limited thereto, and is not limited if it is a biological sample isolated from a patient suspected of having 2019 novel coronavirus infection. A method for isolating total RNA from the sample may use any method known in the art, for example, a phenol extraction method may be used.
본 발명의 일 구현 예에 따른 2019 신종코로나바이러스의 또 다른 검출 방법은,Another detection method of 2019 novel coronavirus according to an embodiment of the present invention is,
2019 신종코로나바이러스감염증 의심 환자로부터 분리된 생물학적 시료를 용해 버퍼로 전처리하는 단계;Pre-treating a biological sample isolated from a patient suspected of 2019 novel coronavirus infection with a lysis buffer;
상기 용해 버퍼로 전처리된 시료를 본 발명의 역전사고리매개등온증폭용 프라이머 세트를 이용하여 역전사고리매개등온증폭 반응으로 표적서열을 증폭하는 단계; 및amplifying the target sequence by a reverse cycle-mediated isothermal amplification reaction using the primer set for reverse cycle-mediated isothermal amplification of the sample pretreated with the lysis buffer; and
상기 증폭 단계의 산물을 검출하는 단계;를 포함할 수 있으나, 이에 제한되지 않는다.Detecting the product of the amplification step; may include, but is not limited thereto.
본 발명의 검출 방법에 따른 시료(예컨대, 코 면봉 시료 또는 분리된 인간 혈액)를 용해 버퍼로 전처리하는 단계는, 바람직하게는 시료와 용해 버퍼를 1:9의 부피비로 혼합하고 5분 이내에 수행하는 것일 수 있으나, 이에 제한되지 않는다. 상기 용해 버퍼는 바람직하게는 0.1~1.5 %(v/v)의 Triton X-100을 포함하는, pH 6.0~7.0의 30~500 mM Tris일 수 있고, 더욱 바람직하게는 0.8~1.2 %(v/v)의 Triton X-100을 포함하는, pH 6.5의 300~500 mM Tris일 수 있으며, 더 더욱 바람직하게는 1 %(v/v)의 Triton X-100을 포함하는, pH 6.5의 400 mM Tris일 수 있으나, 이에 제한되지 않는다.The step of pretreating the sample (eg, nasal swab sample or isolated human blood) with the lysis buffer according to the detection method of the present invention is preferably performed within 5 minutes after mixing the sample and the lysis buffer in a volume ratio of 1:9. may be, but is not limited thereto. The lysis buffer may be 30-500 mM Tris of pH 6.0-7.0, preferably containing 0.1-1.5% (v/v) of Triton X-100, more preferably 0.8-1.2% (v/v) It may be 300-500 mM Tris at pH 6.5, including Triton X-100 of v), and even more preferably 400 mM Tris at pH 6.5, containing 1% (v/v) Triton X-100. may be, but is not limited thereto.
본 발명의 2019 신종코로나바이러스 검출 방법에 있어서, 상기 등온증폭 반응은 62℃ 내지 68℃에서 20분 내지 50분 동안 수행될 수 있고, 바람직하게는 65℃에서 20분 내지 50분 동안 수행되는 것일 수 있으나, 이에 제한되지 않는다.In the 2019 novel coronavirus detection method of the present invention, the isothermal amplification reaction may be performed at 62 ° C. to 68 ° C. for 20 minutes to 50 minutes, and preferably at 65 ° C. for 20 minutes to 50 minutes. However, it is not limited thereto.
본 발명의 2019 신종코로나바이러스 검출 방법은 상기 역전사고리매개등온증폭 단계의 산물을 검출하는 단계를 포함한다. 상기 증폭 산물의 검출은 SYBR Green I을 이용한 형광 측정, 페놀 레드와 같은 지시약의 색상 변화, 탁색도, 침전물 생성, DNA laddering, 모세관 전기영동, DNA 칩, 겔 전기영동, 방사성 측정, 또는 인광 측정을 통해 수행될 수 있고, 바람직하게는 SYBR Green I을 이용한 형광 측정, 탁색도, DNA laddering일 수 있으나, 이에 제한되지 않는다.The 2019 novel coronavirus detection method of the present invention includes detecting the product of the reverse thought-mediated isothermal amplification step. The detection of the amplification product is fluorescence measurement using SYBR Green I, color change of an indicator such as phenol red, turbidity, precipitate formation, DNA laddering, capillary electrophoresis, DNA chip, gel electrophoresis, radiometric measurement, or phosphorescence measurement. It may be performed through, preferably, fluorescence measurement using SYBR Green I, turbidity, and DNA laddering, but is not limited thereto.
또한, 반응액 중 형광염료 등을 실시간으로 검출할 수 있는 장비를 이용하여 별도의 전기영동(electrophoresis)이나 반응 후의 SYBR Green I 추가 없이 실시간으로 등온증폭을 확인하고, annealing peak를 통해서 표적서열의 증폭 여부를 확인할 수도 있다.In addition, using equipment that can detect fluorescent dyes in the reaction solution in real time, check isothermal amplification in real time without additional electrophoresis or addition of SYBR Green I after the reaction, and amplification of the target sequence through annealing peak You can also check whether
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.
실시예 1. 2019 신종코로나바이러스 검출을 위한 LAMP 프라이머 세트 설계Example 1. Design of LAMP primer set for 2019 novel coronavirus detection
2019 신종코로나바이러스(2019 novel Coronavirus)의 검출을 위한 고리매개등온증폭반응(LAMP)용 프라이머 세트를 제작하기 위해, GenBank accession no. MN908947.3인 Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, complete genome에서 뉴클레오캡시드 단백질을 코딩하는 유전자(28,274~29,533, 서열번호 7)의 정보를 확보하였다. LAMP에 사용할 프라이머는 OptiGene사의 LAMP Design software를 이용하여 설계하였으며, 최종 설계된 2019 신종코로나바이러스 검출을 위한 LAMP 프라이머는 서열번호 7의 염기서열로 이루어진 뉴클레오캡시드 단백질 코딩 유전자 서열에서 401~700번째 염기서열 부위를 표적으로 하며, 각 LAMP 프라이머 서열의 위치는 도 1에 나타낸 바와 같다. 상기 서열번호 7의 염기서열로 이루어진 뉴클레오캡시드 단백질 코딩 유전자 서열에서 401~700번째 염기서열 부위를 다양한 2019 코로나바이러스 염기서열(GenBank accession no: LC521925, LR757995, LR757996, LR757997, LR757998, MN938384, MN975262, MN988668, MN988669, MN985325, MN988713, MN994467, MN994468, MN997409 및 MT007544)과 정렬해본 결과, 본 발명에 따른 서열번호 1 내지 6의 LAMP 프라이머 세트가 표적하는 위치는 보존된 영역임을 확인할 수 있었다.To produce a primer set for a ring-mediated isothermal amplification reaction (LAMP) for the detection of the 2019 novel Coronavirus, GenBank accession no. Information on the gene (28,274-29,533, SEQ ID NO: 7) encoding the nucleocapsid protein was obtained from the MN908947.3, Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, complete genome. The primer to be used for LAMP was designed using OptiGene's LAMP Design software, and the finally designed LAMP primer for 2019 novel coronavirus detection is nucleotide sequence 401 to 700 in the nucleocapsid protein coding gene sequence consisting of the nucleotide sequence of SEQ ID NO: 7 The site is targeted, and the position of each LAMP primer sequence is as shown in FIG. 1 . In the nucleocapsid protein-coding gene sequence consisting of the nucleotide sequence of SEQ ID NO: 7, the 401 to 700 nucleotide sequence region was converted to various 2019 coronavirus nucleotide sequences (GenBank accession no: LC521925, LR757995, LR757996, LR757997, LR757998, MN938384, MN975262, As a result of alignment with MN988668, MN988669, MN985325, MN988713, MN994467, MN994468, MN997409 and MT007544), it was confirmed that the positions targeted by the LAMP primer sets of SEQ ID NOs: 1 to 6 according to the present invention are conserved regions.
또한, 이전의 연구에 의하면 2019 신종코로나바이러스의 뉴클레오캡시드 단백질을 코딩하는 유전자( N 유전자)를 표적으로 했을 때 다른 인체 전염 코로나바이러스들 (HCoV 229E, HCoV OC43, HCoV HKU1, HCoV NL63 및 MERS-CoV)은 검출되지 않는 결과들이 확인되었고(Corman Victor M. et al., 2020 Euro Surveill. 25(3):pii=2000045), 가장 비슷한 서열인 SARS-CoV의 경우 염기서열을 정렬해본 결과, 본 발명의 F3(서열번호 1), B3(서열번호 2) 및 BIP 프라이머(서열번호 3)가 표적하는 염기서열과 정보가 상이하여(도 5) LAMP의 덤벨구조 형성이 되지 않아 정상적인 LAMP 반응이 되지 않을 것으로 예상되었다.In addition, according to previous studies, when the gene encoding the nucleocapsid protein ( N gene) of the 2019 novel coronavirus was targeted, other human infectious coronaviruses (HCoV 229E, HCoV OC43, HCoV HKU1, HCoV NL63 and MERS- CoV) was confirmed (Corman Victor M. et al ., 2020 Euro Surveill. 25(3):pii=2000045), and in the case of SARS-CoV, which is the most similar sequence, as a result of aligning the nucleotide sequence, this F3 (SEQ ID NO: 1), B3 (SEQ ID NO: 2) and BIP primer (SEQ ID NO: 3) of the invention are different from the target nucleotide sequence and information (FIG. 5) was not expected
실시예 2. 2019 신종코로나바이러스Example 2. 2019 Novel Coronavirus 검출능 분석Detectability analysis
제작된 LAMP 프라이머를 이용한 2019 신종코로나바이러스의 검출능을 분석하기 위해, 임상검체를 이용한 RT-LAMP를 수행하였다. 2019 신종코로나바이러스 감염 임상검체는 국군 수도병원으로부터 제공받아 사용하였으며, 본 발명의 LMAP 프라이머 세트가 2019 신종코로나바이러스에 특이적으로 결합하는지를 확인하기 위해, 유사한 임상 증상(기침 및/또는 발열)을 유발하는 아데노바이러스 55형( Adenovirus type 55), 헤모필루스 인플루엔자( Haemophilus Influenzae), 스트렙토코커스 뉴모니에( Streptococcus pneumoniae), 마이코플라즈마 뉴모니에( Mycoplasma pneumonia), 중증열성혈소판감소증후군 바이러스(Severe fever with thrombocytopenia syndrome virus, SFTS virus), 말라리아, 쯔쯔가무시병(Scrub typhus) 감염 임상검체(호흡기 검체)를 육군훈련소로부터 제공받아 비교군으로 사용하였다.In order to analyze the detection ability of the 2019 novel coronavirus using the prepared LAMP primer, RT-LAMP using a clinical sample was performed. The 2019 novel coronavirus infection clinical specimen was provided and used by the Armed Forces Capital Hospital, and to confirm whether the LMAP primer set of the present invention specifically binds to the 2019 novel coronavirus, similar clinical symptoms (cough and/or fever) were induced. Adenovirus type 55, Haemophilus Influenzae , Streptococcus pneumoniae , Mycoplasma pneumoniae , Severe fever with thrombocytopenia syndrome virus, SFTS virus), malaria, and Scrub typhus infection clinical specimens (respiratory specimens) were provided by the Army Training Center and used as a comparison group.
RT-LAMP(Reverse transcriptional Loop-mediated Isothermal Amplification) 반응은 주형 RNA를 2㎕, Bst 폴리머라제 1㎕ (8U), 10×buffer 2.5㎕, 25mM dNTP 1.5㎕, 10mM MgSO 4 1㎕, 5M betaine 5㎕, Reverse Transcriptase 0.1㎕ (10U), 6개의 각 프라이머(F3 및 B3는 각각 5pmol, FIP 및 BIP는 각각 40pmol, LoopF 및 LoopB는 각각 10pmol) 각 1㎕씩, 25×SYBR Green I 1㎕ 및 증류수(up to ~25㎕)를 포함하는 총 25㎕의 반응액에서 수행하였다. 실시간으로 등온증폭 산물의 결과를 확인하기 위해, BioRad CFX 96 Real time PCR 기기 또는 휴대용 Isothermal Fluorescence PCR 기기인 Gene-8C (ALLSHENG)를 사용하여 65℃, 20분간 등온증폭 반응을 수행하였다. 형광 신호는 매 30초마다 측정하였다.RT-LAMP (Reverse Transcriptional Loop-mediated Isothermal Amplification) reaction consisted of 2 μl of template RNA, 1 μl of Bst polymerase (8U), 2.5 μl of 10×buffer, 1.5 μl of 25mM dNTP, 1 μl of 10mM MgSO 4 , 5 μl of 5M betaine. , Reverse Transcriptase 0.1 μl (10U), 6 primers (5 pmol each for F3 and B3, 40 pmol each for FIP and BIP, 10 pmol each for LoopF and LoopB) 1 μl each, 25×SYBR Green I 1 μl and distilled water ( up to ~25 μl) in a total of 25 μl of the reaction solution. In order to confirm the result of the isothermal amplification product in real time, an isothermal amplification reaction was performed at 65° C. for 20 minutes using a BioRad CFX 96 Real time PCR device or a portable Isothermal Fluorescence PCR device, Gene-8C (ALLSHENG). Fluorescence signals were measured every 30 seconds.
Real time PCR 기기에서 수행된 RT-LAMP 결과, 22 cycles에서부터 2019 신종코로나바이러스 감염 시료에서만 증폭이 일어나기 시작해 30 cycles에서는 2019 신종코로나바이러스 감염 시료가 특이적으로 검출되고 있음을 확인할 수 있었다(도 2). 또한, 휴대용 등온증폭 기기에서 수행된 RT-LAMP 결과, 반응 시간 16분 경과 후부터 2019 신종코로나바이러스 감염 시료에서만 증폭 산물이 확인되었고, 반응이 종료되는 20분까지 헤모필루스 인플루엔자( H. Influenzae), 스트렙토코커스 뉴모니에( S. pneumoniae) 및 마이코플라즈마 뉴모니에( M. pneumonia) 시료에서는 증폭산물이 전혀 확인되지 않음을 알 수 있었다(도 3). 즉, 본 발명에 따른 RT-LAMP 반응은 2019 신종코로나바이러스를 특이적으로 검출할 수 있는 방법임을 알 수 있었다.As a result of RT-LAMP performed on a real-time PCR device, it was confirmed that amplification started only in the 2019 novel coronavirus-infected sample from 22 cycles, and that the 2019 novel coronavirus-infected sample was specifically detected at 30 cycles (Fig. 2) . In addition, as a result of the RT-LAMP performed on a portable isothermal amplification device, the amplification product was confirmed only in the 2019 novel coronavirus-infected sample after 16 minutes of reaction time, and H. Influenzae , Streptococcus, until 20 minutes when the reaction was terminated. Pneumoniae ( S. pneumoniae ) and Mycoplasma pneumoniae ( M. pneumonia ) It was found that no amplification products were identified in the samples ( FIG. 3 ). That is, it was found that the RT-LAMP reaction according to the present invention is a method capable of specifically detecting the 2019 novel coronavirus.
실시예 3. Direct RT-LAMP 분석능 평가Example 3. Direct RT-LAMP analysis performance evaluation
코 면봉법(nasal swab)으로 획득한 시료 50㎕를 본 발명자가 개발한 용해 버퍼(400mM Tris, pH 6.5, 1% Triton X-100) 450㎕와 혼합한 후 볼텍싱을 통해 섞어준 후, 상기 혼합액 중 2㎕를 취하여 LAMP 프리믹스 (Bst 폴리머라제 1㎕ (8U), 10×buffer 2.5㎕, 25mM dNTPs 1.5㎕, 10mM MgSO 4 1㎕, 5M betaine 5㎕), 6개의 프라이머(F3 및 B3는 각각 5pmol, FIP 및 BIP는 각각 40pmol, LoopF 및 LoopB는 각각 10pmol) 각 1㎕씩, 25X SYBR Green I 1㎕ 및 증류수(up to ~25㎕)를 첨가하고, Isothermal Fluorescence PCR, Gene-8C (ALLSHENG, 중국)를 이용하여 65℃, 50분간 등온증폭 반응을 수행하였다. 형광 신호는 매 15초마다 측정하였다.After mixing 50 μl of the sample obtained by nasal swab with 450 μl of the lysis buffer (400 mM Tris, pH 6.5, 1% Triton X-100) developed by the present inventors and mixing through vortexing, the 2 μl of the mixture was taken and LAMP premix (Bst polymerase 1 μl (8U), 10×buffer 2.5 μl, 25 mM dNTPs 1.5 μl, 10 mM MgSO 4 1 μl, 5M betaine 5 μl), 6 primers (F3 and B3 were each 5 pmol, 40 pmol each for FIP and BIP, 10 pmol each for LoopF and LoopB) 1 μl each, 25X SYBR Green I 1 μl and distilled water (up to ~ 25 μl) were added, and Isothermal Fluorescence PCR, Gene-8C (ALLSHENG, An isothermal amplification reaction was carried out at 65° C. for 50 minutes using a Chinese). The fluorescence signal was measured every 15 seconds.
그 결과, 도 3과 비교하여 증폭 산물이 확인되는 검출 시간이 지연되긴 하였으나, 별도의 핵산 추출 및 정제 단계가 포함되지 않은 Direct RT-LAMP 반응의 결과에서도 반응 시간 42분 이후부터 2019 신종코로나바이러스 감염 시료에서만 증폭 산물이 확인되고, 헤모필루스 인플루엔자( H. Influenzae) 및 마이코플라즈마 뉴모니에( M. pneumonia) 감염 시료에서는 반응 종료 시점까지 증폭 산물이 확인되지 않아(도 4), 본 발명에 따른 Direct RT-LAMP을 통해 2019 신종코로나바이러스를 정확하게 검출할 수 있음을 알 수 있었다. 이상의 결과를 통해 본 발명의 프라이머 세트 및 이를 이용한 2019 신종코로나바이러스 검출 방법은 2019 신종코로나바이러스(우한 폐렴) 감염 진단에 매우 유용하게 활용될 수 있을 것으로 예상되었다.As a result, although the detection time at which the amplification product is confirmed was delayed compared to FIG. 3, even in the result of the Direct RT-LAMP reaction that does not include a separate nucleic acid extraction and purification step, the 2019 novel coronavirus infection started after 42 minutes. The amplification product was confirmed only in the sample, and in the sample infected with Haemophilus influenzae ( H. Influenzae ) and Mycoplasma pneumoniae ( M. pneumonia ), the amplification product was not confirmed until the end of the reaction ( FIG. 4 ), Direct RT according to the present invention -LAMP was able to accurately detect the 2019 novel coronavirus. Based on the above results, it was expected that the primer set of the present invention and the 2019 novel coronavirus detection method using the same could be very usefully utilized for diagnosing the 2019 novel coronavirus (Wuhan pneumonia) infection.
실시예 4. 2019 신종코로나바이러스 검출을 위한 LAMP 프라이머 세트의 특이도 및 민감도 분석Example 4. Analysis of Specificity and Sensitivity of LAMP Primer Set for Detection of 2019 Novel Coronavirus
본 발명자는 2019 신종코로나바이러스(2019-nCoV), SARS-CoV (Severe acute respiratory syndrome coronavirus) 및 MERS-CoV (Middle East respiratory syndrome coronavirus)의 N 유전자 염기서열을 각각 합성한 후, 해당 서열을 주형으로 본 발명의 프라이머 세트의 특이성을 검증하였다. The present inventor synthesized the N gene sequences of 2019 novel coronavirus (2019-nCoV), SARS-CoV (Severe acute respiratory syndrome coronavirus) and MERS-CoV (Middle East respiratory syndrome coronavirus), respectively, and then used the sequence as a template The specificity of the primer set of the present invention was verified.
주형으로 사용한 SARS-CoV 및 MERS-CoV의 N 유전자 염기서열은 각각 NCBI accession no. MK062184.1 서열의 28,719~28,961번째 및 NCBI accession no. MN723544.1 서열의 28,982~29,232번째 서열을 참고하여 바이오닉스(주)에 합성의뢰하여 준비하였으며, 2019-nCoV의 N 유전자 주형은 도 1에 개시된 서열과 동일하게 준비하였다. LAMP 반응은 주형 cDNA(1 fg)를 5㎕, Bst 폴리머라제 1㎕ (8U), 10x buffer 2.5㎕, 25mM dNTP 1.5㎕, 10mM MgSO 4 1㎕, 5M betaine 5㎕, 6개의 각 프라이머(F3 및 B3는 각각 5pmol, FIP 및 BIP는 각각 40pmol, LoopF 및 LoopB는 각각 10pmol) 각 1㎕씩, 25x SYBR Green I 1㎕ 및 증류수(up to ~25㎕)를 포함하는 총 25㎕의 반응액을 조성하여 BioRad CFX 96 Real time PCR 기기를 사용하여 65℃에서 30분간 등온증폭반응을 수행하였다. 그 결과, 도 6에서와 같이 본 발명의 프라이머 세트는 2019 신종코로나바이러스의 N 유전자를 포함하는 시료에서는 22 cycles 시점부터 형광 시그널이 증폭되는 것이 확인되었고, SARS-CoV 및 MERS-CoV의 N 유전자를 포함하는 시료에서는 반응 종료 시점까지 형광 시그널이 확인되지 않아 증폭 반응이 일어나지 않았음을 관찰할 수 있었다. 이를 통해, 본 발명에서 제작한 서열번호 1 내지 6의 올리고뉴클레오티드 프라이머를 포함하는 RT-LAMP용 프라이머 세트는 2019 신종코로나바이러스만을 특이적으로 검출할 수 있음을 확인할 수 있었다. The N gene sequences of SARS-CoV and MERS-CoV used as templates are, respectively, NCBI accession no. 28,719-28,961 of the sequence MK062184.1 and NCBI accession no. By referring to the 28,982~29,232th sequence of the MN723544.1 sequence, it was prepared by requesting synthesis from Bionics Co., Ltd., and the N gene template of 2019-nCoV was prepared in the same manner as the sequence disclosed in FIG. 1 . For LAMP reaction, 5 μl of template cDNA (1 fg), 1 μl of Bst polymerase (8U), 2.5 μl of 10x buffer, 1.5 μl of 25 mM dNTP, 1 μl of 10 mM MgSO 4 , 5 μl of 5M betaine, 6 primers (F3 and B3 is 5 pmol each, FIP and BIP are 40 pmol each, LoopF and LoopB are 10 pmol each) 1 μl each, 25x SYBR Green I 1 μl and distilled water (up to ~ 25 μl) A total of 25 μl of reaction solution is prepared Thus, an isothermal amplification reaction was performed at 65° C. for 30 minutes using a BioRad CFX 96 Real time PCR device. As a result, the primer sets of the present invention, as shown in 6 has been confirmed that the fluorescent signal amplification from 22 cycles time for a sample containing the N gene of the 2019 swine coronavirus, the N gene of SARS-CoV and MERS-CoV In the containing sample, it was observed that the amplification reaction did not occur because the fluorescence signal was not confirmed until the reaction was terminated. Through this, it was confirmed that the primer set for RT-LAMP including the oligonucleotide primers of SEQ ID NOs: 1 to 6 prepared in the present invention can specifically detect only the 2019 novel coronavirus.
또한, 실시간 LAMP 반응을 통해 본 발명에 따른 프라이머 세트의 검출 한계(limit of detection, LOD)를 확인해 본 결과, 본 발명의 LAMP 프라이머 세트의 검출 한계는 10 0 copy 즉, 1 copy로 확인되어 민감도가 매우 우수한 것을 알 수 있었다(도 7).In addition, the detection limit of the primer sets (limit of detection, LOD) to confirm the results, the detection limit of LAMP primer set of the present invention is identified as 10 0 copy i.e., 1 copy sensitivity in accordance with the present invention provides real-time LAMP reaction It was found that it was very good (FIG. 7).

Claims (8)

  1. 서열번호 1 내지 6의 올리고뉴클레오티드 프라이머를 포함하는 2019 신종코로나바이러스(2019 novel Coronavirus)를 검출하기 위한 역전사고리매개등온증폭(Reverse transcriptional Loop-mediated isothermal amplification)용 프라이머 세트.A primer set for Reverse Transcriptional Loop-mediated isothermal amplification for detecting the 2019 novel Coronavirus comprising the oligonucleotide primers of SEQ ID NOs: 1 to 6.
  2. 제1항에 따른 역전사고리매개등온증폭용 프라이머 세트를 포함하는 2019 신종코로나바이러스(2019 novel Coronavirus) 검출용 조성물.A composition for detecting the 2019 novel coronavirus comprising the primer set for reverse cycle mediated isothermal amplification according to claim 1.
  3. 제1항에 따른 역전사고리매개등온증폭용 프라이머 세트 및 고리매개등온증폭 반응을 수행하기 위한 시약을 포함하는 2019 신종코로나바이러스(2019 novel Coronavirus) 검출용 키트.A kit for detecting 2019 novel Coronavirus, comprising a primer set for reverse cycle-mediated isothermal amplification according to claim 1 and a reagent for performing a ring-mediated isothermal amplification reaction.
  4. 제3항에 있어서, 상기 키트는 0.1~1.5 %(v/v)의 Triton X-100을 포함하는, pH 6.0~7.0의 30~500 mM Tris로 이루어진 용해 버퍼를 추가로 포함하는 것을 특징으로 하는 2019 신종코로나바이러스 검출용 키트.The method of claim 3, wherein the kit further comprises a lysis buffer consisting of 30-500 mM Tris of pH 6.0-7.0, containing 0.1-1.5% (v/v) Triton X-100. 2019 novel coronavirus detection kit.
  5. 2019 신종코로나바이러스감염증 의심 환자로부터 분리된 생물학적 시료로부터 총 RNA를 분리하는 단계;isolating total RNA from a biological sample isolated from a patient suspected of having 2019 novel coronavirus infection;
    상기 분리된 총 RNA를 주형으로 하고, 제1항의 역전사고리매개등온증폭용 프라이머 세트를 이용하여 역전사고리매개등온증폭(Reverse transcriptional Loop-mediated Isothermal Amplification) 반응으로 표적서열을 증폭하는 단계; 및Amplifying the target sequence by using the isolated total RNA as a template and using the primer set for reverse transcriptional loop-mediated isothermal amplification of claim 1; and
    상기 증폭 단계의 산물을 검출하는 단계;를 포함하는 2019 신종코로나바이러스(2019 novel Coronavirus)를 검출하는 방법.Detecting the product of the amplification step; A method of detecting a 2019 novel coronavirus comprising a.
  6. 2019 신종코로나바이러스감염증 의심 환자로부터 분리된 생물학적 시료를 용해 버퍼로 전처리하는 단계;Pre-treating a biological sample isolated from a patient suspected of 2019 novel coronavirus infection with a lysis buffer;
    상기 용해 버퍼로 전처리된 시료를 제1항의 역전사고리매개등온증폭용 프라이머 세트를 이용하여 역전사고리매개등온증폭(Reverse transcriptional Loop-mediated Isothermal Amplification) 반응으로 표적서열을 증폭하는 단계; 및Amplifying the target sequence in a reverse transcriptional loop-mediated isothermal amplification reaction using the primer set for reverse cycle-mediated isothermal amplification of the sample pretreated with the lysis buffer of claim 1; and
    상기 증폭 단계의 산물을 검출하는 단계;를 포함하는 2019 신종코로나바이러스(2019 novel Coronavirus)를 검출하는 방법.Detecting the product of the amplification step; A method of detecting a 2019 novel coronavirus comprising a.
  7. 제6항에 있어서, 상기 용해 버퍼는 0.1~1.5 %(v/v)의 Triton X-100을 포함하는, pH 6.0~7.0의 30~500 mM Tris로 이루어진 것을 특징으로 하는 2019 신종코로나바이러스(2019 novel Coronavirus)를 검출하는 방법.The 2019 novel coronavirus (2019) according to claim 6, wherein the lysis buffer is composed of 30-500 mM Tris with a pH of 6.0-7.0, containing 0.1-1.5% (v/v) of Triton X-100. How to detect novel Coronavirus).
  8. 제5항 또는 제6항에 있어서, 상기 증폭 산물의 검출은 형광발색, 탁색도, 스핀다운에 의한 침전물 생성 또는 전기영동을 통해 수행되는 것을 특징으로 하는 2019 신종코로나바이러스를 검출하는 방법.The method according to claim 5 or 6, wherein the detection of the amplification product is performed through fluorescence, turbidity, formation of a precipitate by spin-down, or electrophoresis.
PCT/KR2020/009285 2020-02-11 2020-07-15 Lamp composition for detecting 2019 novel coronavirus, and use thereof WO2021162183A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020200016377A KR102109196B1 (en) 2020-02-11 2020-02-11 LAMP composition for detecting 2019 novel Coronavirus and uses thereof
KR10-2020-0016377 2020-02-11

Publications (1)

Publication Number Publication Date
WO2021162183A1 true WO2021162183A1 (en) 2021-08-19

Family

ID=70729229

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2020/009285 WO2021162183A1 (en) 2020-02-11 2020-07-15 Lamp composition for detecting 2019 novel coronavirus, and use thereof

Country Status (2)

Country Link
KR (1) KR102109196B1 (en)
WO (1) WO2021162183A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113774169A (en) * 2021-09-18 2021-12-10 北京艾瑞克阳医疗科技有限公司 2019 novel coronavirus delta variant nucleic acid detection reagent, kit and detection method

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102109196B1 (en) * 2020-02-11 2020-05-11 대한민국 LAMP composition for detecting 2019 novel Coronavirus and uses thereof
WO2021231706A1 (en) * 2020-05-13 2021-11-18 The Trustees Of Columbia University In The City Of New York Methods and compositions for rapid testing for a target nucleic acid including viral nucleic acid
WO2021245708A1 (en) * 2020-06-05 2021-12-09 Indian Council Of Medical Research A rapid and sensitive method for detecting sars-cov-2
KR20220005219A (en) * 2020-07-06 2022-01-13 주식회사 시선바이오머티리얼스 Qurantine System for High-Lisk Contagious Disease Using Isothermal Amplification Based Molecular Diagnostics
KR102236568B1 (en) 2020-07-07 2021-04-19 주식회사 쉐어플러스 The uncontact specimen collection system to use smart device
KR102264483B1 (en) 2020-07-07 2021-06-17 주식회사 쉐어플러스 The movable uncontact specimen collection booth
KR102247756B1 (en) 2020-08-20 2021-06-03 주식회사 쉐어플러스 walk-through screening clinic with smart function
KR20220025544A (en) 2020-08-24 2022-03-03 에이펙셀 (주) Antiviral composition having with anti-viral activity of coronavirus and variant virus
KR102392907B1 (en) 2020-08-28 2022-05-02 충북대학교 산학협력단 Composition comprising primer set and pna blocker and using thereof
CN112626266A (en) * 2020-10-27 2021-04-09 山西高等创新研究院 Novel detection primer group for coronavirus SARS-CoV-2 and application thereof
KR102393597B1 (en) 2020-12-31 2022-05-04 진온바이오텍(주) New primer set of Loop-mediated isothermal amplification for detection of 2019 novel Coronavirus and a composition containing the same
KR102621111B1 (en) 2021-03-12 2024-01-03 조선대학교산학협력단 Detection composition of SARS-CoV-2 virus gene and COVID-19 diagnosis method using real-time RT-PCR
KR102643238B1 (en) 2021-03-25 2024-03-05 한양대학교 에리카산학협력단 Primer and probe set for detection and identification of SARS-CoV-2 and mutant thereof
KR102658142B1 (en) 2021-05-18 2024-04-17 정문성 Unmanned inspection apparatus for infectious virus, unmanned inspection system using the unmanned inspection apparatus and control method thereof
KR102402765B1 (en) * 2021-09-02 2022-05-30 대한민국 Multiplex RT-LAMP composition for diagnosis of SARS-CoV-2 infection and uses thereof
KR20230064339A (en) 2021-11-03 2023-05-10 재단법인 대구경북첨단의료산업진흥재단 Loop Mediated Isothermal Amplification (LAMP) primer sets for detecting COVID-19 and use threreof
KR20230138683A (en) * 2022-03-24 2023-10-05 전북대학교산학협력단 Composition for Detecting Target Gene based on cDNA Synthesis using a Ligation Method that does not use Reverse Transcription, and Method for multiple Ligation-Assisted Recombinase Polymerase Amplification

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040074264A (en) * 2003-02-17 2004-08-25 주식회사 인트론바이오테크놀로지 Nucleocapsid gene of infectious bronchitis virus
KR20080012449A (en) * 2006-08-03 2008-02-12 재단법인서울대학교산학협력재단 Diagnostic methods for sars by using nucleocapside or spike protein
JP2012024039A (en) * 2010-07-26 2012-02-09 Toshiba Corp Nucleic acid primer set for lamp amplification for corona virus
KR20180052317A (en) * 2016-11-10 2018-05-18 충북대학교 산학협력단 Primer set for detection of MERS-coronavirus and uses thereof
KR102004951B1 (en) * 2018-12-07 2019-08-01 대한민국 Direct LAMP kit and method for diagnosing malaria infection
KR102109196B1 (en) * 2020-02-11 2020-05-11 대한민국 LAMP composition for detecting 2019 novel Coronavirus and uses thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040074264A (en) * 2003-02-17 2004-08-25 주식회사 인트론바이오테크놀로지 Nucleocapsid gene of infectious bronchitis virus
KR20080012449A (en) * 2006-08-03 2008-02-12 재단법인서울대학교산학협력재단 Diagnostic methods for sars by using nucleocapside or spike protein
JP2012024039A (en) * 2010-07-26 2012-02-09 Toshiba Corp Nucleic acid primer set for lamp amplification for corona virus
KR20180052317A (en) * 2016-11-10 2018-05-18 충북대학교 산학협력단 Primer set for detection of MERS-coronavirus and uses thereof
KR102004951B1 (en) * 2018-12-07 2019-08-01 대한민국 Direct LAMP kit and method for diagnosing malaria infection
KR102109196B1 (en) * 2020-02-11 2020-05-11 대한민국 LAMP composition for detecting 2019 novel Coronavirus and uses thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CERAOLO CARMINE, GIORGI FEDERICO M.: "Genomic variance of the 2019‐nCoV coronavirus", JOURNAL OF MEDICAL VIROLOGY, JOHN WILEY & SONS, INC., US, vol. 92, no. 5, 1 May 2020 (2020-05-01), US, pages 522 - 528, XP055835508, ISSN: 0146-6615, DOI: 10.1002/jmv.25700 *
KIM JIN HWA; KANG MINHEE; PARK EUNKYOUNG; CHUNG DOO RYEON; KIM JIYEON; HWANG EUNG SOO: "A Simple and Multiplex Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of SARS-CoV", BIOCHIP JOURNAL, KOREAN BIOCHIP SOC, SEOUL , SOUTH KOREA, vol. 13, no. 4, 1 January 1900 (1900-01-01), Seoul , South Korea, pages 341 - 351, XP037382102, ISSN: 1976-0280, DOI: 10.1007/s13206-019-3404-3 *
YU FEI; DU LANYING; OJCIUS DAVID M.; PAN CHUNGEN; JIANG SHIBO: "Measures for diagnosing and treating infections by a novel coronavirus responsible for a pneumonia outbreak originating in Wuhan, China", MICROBES AND INFECTION, ELSEVIER, PARIS, FR, vol. 22, no. 2, 1 February 2020 (2020-02-01), FR, pages 74 - 79, XP086085029, ISSN: 1286-4579, DOI: 10.1016/j.micinf.2020.01.003 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113774169A (en) * 2021-09-18 2021-12-10 北京艾瑞克阳医疗科技有限公司 2019 novel coronavirus delta variant nucleic acid detection reagent, kit and detection method

Also Published As

Publication number Publication date
KR102109196B1 (en) 2020-05-11

Similar Documents

Publication Publication Date Title
WO2021162183A1 (en) Lamp composition for detecting 2019 novel coronavirus, and use thereof
CN111004870B (en) Novel coronavirus N gene nucleic acid detection kit
CN111394513A (en) Fluorescent quantitative PCR detection method for novel coronavirus SARS-CoV-2 and application thereof
CN114369688B (en) Compositions, kits, methods and uses for detecting a variant of SARS-CoV-2 Oncuronte
CN113881812B (en) Composition, kit and method for detecting SARS-CoV-2 mutant strain and use thereof
CN113981152B (en) Composition, kit and method for detecting SARS-CoV-2 variant strain and its use
CN113652505B (en) Method and kit for detecting novel coronavirus and VOC-202012/01 mutant strain thereof
KR20210122632A (en) PNA Probe and Primer for Detecting SARS-CoV-2 Causing Covid-19 Using RT-LAMP and Method for Detecting SARS-CoV-2 Infection Using Thereof
WO2021225221A1 (en) Rt-lamp kit and method for diagnosis of coronavirus-19 infection
WO2022050700A1 (en) Human sars-coronavirus-2 universal primer set for whole genome amplification, and diagnostic kit using same
WO2022005255A2 (en) Simultaneous diagnosis method and kit for sars-cov-2 inducing coronavirus disease-19 and sarbecovirus using pna probe
KR20220040834A (en) Multiplex LAMP composition for diagnosis of COVID-19 comprising molecular beacon probe
KR102428504B1 (en) RT-LAMP pirmer set for detecting SARS-CoV-2 omicron variant and uses thereof
KR102076343B1 (en) Composition for detecting adenovirus type 55 using Real-time LAMP and uses thereof
WO2022145658A1 (en) Composition for diagnosing tsutsugamushi disease and kit comprising same
KR102346881B1 (en) A kit for simultaneous diagnosis of coronavirus and influenza virus
EP3885455A1 (en) Method and kit for the detection of sars-cov-2 virus in a sample based on reverse transcription loop-mediated isothermal amplification (rt-lamp)
CN113817870A (en) Primer composition for simultaneously detecting seven respiratory tract-related viruses and application thereof
KR102393597B1 (en) New primer set of Loop-mediated isothermal amplification for detection of 2019 novel Coronavirus and a composition containing the same
CN114262758B (en) Kit for detecting novel coronavirus mutant strain and detection method
KR102402765B1 (en) Multiplex RT-LAMP composition for diagnosis of SARS-CoV-2 infection and uses thereof
CN117551818B (en) Target gene, primer probe combination and kit for detecting respiratory syncytial virus and application
KR102366554B1 (en) RT-LAMP pirmer set for discriminating SARS-CoV-2 delta variant and uses thereof
KR102306941B1 (en) Composition for detecting SARS-CoV-2 viral RNA and diagnosis of COVID-19 based on real time RT-PCR
WO2022010206A1 (en) Quarantine system for high-risk infectious disease using isothermal amplification-based molecular diagnostics

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20918209

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20918209

Country of ref document: EP

Kind code of ref document: A1