WO2022010206A1 - Quarantine system for high-risk infectious disease using isothermal amplification-based molecular diagnostics - Google Patents

Quarantine system for high-risk infectious disease using isothermal amplification-based molecular diagnostics Download PDF

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WO2022010206A1
WO2022010206A1 PCT/KR2021/008506 KR2021008506W WO2022010206A1 WO 2022010206 A1 WO2022010206 A1 WO 2022010206A1 KR 2021008506 W KR2021008506 W KR 2021008506W WO 2022010206 A1 WO2022010206 A1 WO 2022010206A1
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seq
primer
quarantine system
test
covid
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PCT/KR2021/008506
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French (fr)
Korean (ko)
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박성민
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주식회사 시선바이오머티리얼스
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    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the present invention relates to a quarantine and quarantine system for a high-risk infectious disease, Corona 19 virus, and more specifically, Corona 19 quarantine using isothermal amplification-based molecular diagnostics for SARS-CoV-2 detection It relates to the system, especially the quarantine system in airports, ports, large gatherings, and cluster infection areas.
  • Coronaviruses are known to cause a variety of diseases in animals, such as gastrointestinal and respiratory diseases.
  • Human coronaviruses that infect humans are HCoV-229E and HCoV-OC43 discovered in the 1960s, and HCoV-NL63 (2004) and HCoV-HKU1 (2005) discovered after the SARS pandemic, which are generally not associated with upper respiratory tract infections. It is known that it can lead to serious lung disease in immunodeficiency patients. It is reported that the infection rate for coronavirus increases mainly in winter or early spring, and it is known that the proportion of the pathogen that is the cause of the coronavirus among adult cold patients is quite high.
  • SARS-CoV which causes Severe Acute Respiratory Syndrome (SARS)
  • WHO World Health Organization
  • HCV-EMC Middle East Respiratory Syndrome Coronavirus
  • Coronavirus Infectious Disease-19 is a viral respiratory disease that occurred in Wuhan, China in December 2019. It is also called 'Wuhan pneumonia', 'novel coronavirus infection', 'corona 19'. It is an epidemic disease caused by the novel coronavirus, which is transmitted through the respiratory tract, and shows strong contagious characteristics in the early stages of infection with few symptoms. After infection, symptoms such as sore throat, high fever, cough, and shortness of breath develop into pneumonia. After it spread worldwide in March 2020, the World Health Organization declared the disease a pandemic.
  • Coronavirus Infectious Disease-19 (Covid-19) is mainly transmitted through the respiratory tract. When infected, the virus invades the lungs, causing symptoms such as high fever, coughing, and shortness of breath, and after showing symptoms similar to pneumonia, in severe cases, the alveoli are damaged, leading to death from respiratory failure.
  • the incubation period is 3 to 7 days, but can last up to 14 days. On January 30, 2020, China announced that there were cases where the incubation period was extended to 23 days. It has been reported that COVID-19 is transmitted even during the incubation period when symptoms do not appear.
  • SARS-CoV-2 which currently causes novel coronavirus infection (COVID-19), is basically diagnosed by 'conventional PCR' and sequencing analysis.
  • the pan-coronavirus test method first tests for the presence or absence of all coronaviruses, including the novel coronavirus, and if it is negative, it means that it is not a coronavirus infection. If it is positive, it is determined whether it is another coronavirus causing a cold or a novel coronavirus through gene sequencing. Therefore, it takes 1 to 2 days for diagnosis.
  • the Korea Centers for Disease Control and Prevention (KCDC) is carrying out special quarantine for domestic and foreign nationals entering Korea in order to block the entry of people infected with COVID-19 from overseas.
  • KCDC Korea Centers for Disease Control and Prevention
  • the special entry procedure involves filling out a health status questionnaire and special quarantine report on board the aircraft before entering the country, temperature check at the arrival hall quarantine, submitting a health status questionnaire, and checking for symptoms. quarantined, collecting samples and conducting diagnostic tests.
  • a 'mobile self-diagnosis app' is installed to monitor health conditions such as fever and cough for 14 days after entry.
  • An object of the present invention is a quarantine system using the AQ-TOP TM COVID-19 Rapid kit to quickly screen a person infected with or suspected of being infected with the COVID-19 virus, specifically a new airport, port named 4S (Seasun Smart Shield System) , to provide a quarantine system for large gatherings and cluster infection areas.
  • 4S Seasun Smart Shield System
  • the present invention provides a COVID-19 quarantine system using AQ-TOP TM COVID-19 Rapid kit for detecting SARS-CoV-2, in particular, a quarantine system for airports, ports, large gatherings, mass infection areas, etc. do.
  • 1 is a schematic representation of the current state of the existing COVID-19 test product.
  • FIG. 3 shows a pulling test method in the quarantine system according to the present invention.
  • FIG. 4 shows a single test method in the quarantine system according to the present invention.
  • FIG 5 shows an emergency test method in the quarantine system according to the present invention.
  • a quarantine system that can perform large-scale quarantine faster and faster than the existing method has been developed, and a primer pair capable of amplifying the ORF1ab gene and N gene of SARS-CoV-2 virus, and the primer pair amplified by the
  • This is a gene amplification method that uses a PNA probe that can detect products with high sensitivity and specificity.
  • a quarantine system applied with the "Seasun Smart Shield System (4S)" was developed.
  • the present invention relates to a COVID-19 quarantine system using AQ-TOPTM COVID-19 Rapid Kit for SARS-CoV-2 detection.
  • the present invention is a COVID-19 quarantine system using a molecular diagnostic method through gene amplification, and in particular, it is preferable to use an isothermal amplification-based molecular diagnostic method, but is not limited thereto.
  • the airport quarantine system consists of entry inspection, departure inspection, and emergency inspection. As an inspection, many inspections must be performed in a short time.
  • the pooling test (pooling test), the single test (single test) and the emergency test (emergency test) are all identification steps; sampling step; virus isolation step; And it may be characterized in that it proceeds to the diagnosis / analysis step (FIG. 2).
  • the pooling test, the single test, and the emergency test all require an identification step, and all require a clinical examination diagnosis consent form and an individual identification tag, and the clinical examination
  • a clinical examination diagnosis consent form and an individual identification tag For example, in the case of entering from the airport, it is desirable to collect the consent form after distributing it on board, and in the case of departure inspection, it is desirable to distribute and collect it in the dedicated waiting room.
  • the individual identification tag may be performed by attaching a sticker to a bracelet or identification card (passport, resident registration card, driver's license, etc.) (FIG. 3).
  • the pooling test is characterized in that it is carried out in a manner in which three or four samples are put into one tube and tested.
  • the pooling test is performed with a Big Screening system in which 3 to 4 specimens are placed in one VTM tube and designated as one group for inspection. It can test 760 people in an hour, and additional detailed tests are required to confirm the infection.
  • the Big Screening system uses the Top TM Virus Collection kit (Sisun Biomaterials, Korea), and after collecting the virus, the Top TM Vrial DNA/RNA Exraction Kit (Sissun Biomaterials, Korea) is used. was used to isolate virus DNA and RNA, and SARS-CoV-2 was detected using AQ-TOP TM CIVID-19 Rapid Kit (Sisun Biomaterials, Korea), and gene amplification was performed by CFX96 (Bio-Rad). Alternatively, amplification is performed using an ABI7500 (ABI) instrument (Table 1).
  • the sample odor of the pooling test upon entry at the airport is 37.5 ° C or less according to the fever standard (37.5 ° C), "Oral swab”, 37.5 ° C or more Sampling is carried out with a “Nasal swab”, and a medical professional and flight attendant accompany them to collect a sample at the Connection Bridge, and collect samples by grouping 1 row-1 for each seat in the cabin (4 people/tube) It is preferable to proceed with
  • the "virus isolation step” proceeds with an automatic magnetic bead system (Automation Magnetic bead System), and it is preferable that it takes about 20 minutes to proceed with the 48 test.
  • an automatic magnetic bead system Automation Magnetic bead System
  • the "diagnosis/analysis step" of the pooling test is performed using AQ-TOPTM COVID-19 Rapid Kit (Sightsun Biomaterials, Korea) and CFX96 or CFX384 equipment (Bio-Rad or ABI). Apply to proceed with PCR amplification. It is preferable that it takes about 30 minutes.
  • the single test is characterized in that the test is performed by putting one specimen in one tube as a single test (FIG. 4).
  • a single test enables a quick result report and a smooth end of a situation through a quick and accurate test.
  • the single test is carried out with the Urgent Screening system in which a single test system is applied 1:1 by putting one specimen into one VTM tube, and accurate diagnosis is possible in a short time, Up to 64 people can be tested in 30 minutes.
  • the Urgent Screening system uses the Top TM Virus Collection kit (Siksun Biomaterials, Korea), and after collecting the virus, the Top TM Vrial DNA/RNA Exraction Kit (Sissun Biomaterials, Korea) is used. isolates the DNA and RNA of the virus, and detects SARS-CoV-2 using the AQ-TOP TM COVID-19 Rapid Kit (Sisun Biomaterials, Korea), s, Korea) and PCR amplification takes about 15 minutes (Table 2).
  • 37.5°C or less passes the single test, and 37.5°C or more is sampled with a “Nasal swab” and collect samples in the waiting room with specialized medical personnel.
  • the "virus isolation step” proceeds with an automatic magnetic bead system (Automation Magnetic bead System), and it is preferable that it takes about 20 minutes to proceed with the 48 test.
  • an automatic magnetic bead system Automation Magnetic bead System
  • the "diagnosis/analysis step" of the single test is PCR by applying AQ-TOPTM COVID-19 Rapid Kit (SEASUN) and Sesun Bio isothermal amplification equipment (Seasun Biomaterials, Korea) It is preferable that the amplification process takes about 15 minutes.
  • the emergency test is characterized in that the test is performed by putting one specimen in one tube as a single test (FIG. 5).
  • the emergency test prevents/isolates the spread of infection in indoor facilities and areas in advance so that the examination for infectious diseases can be carried out smoothly with a rapid and accurate quarantine system, Subjects are tested primarily, and groups that are positive for infectious diseases through pooling test are targeted.
  • the urgent test is carried out by an urgent screening system that applies a single test system that is conducted 1:1 by putting one specimen into one VTM tube, and accurate diagnosis is possible in a short time, Up to 64 people can be tested in 30 minutes.
  • the Urgent Screening system uses the Top TM Virus Collection kit (Siksun Biomaterials, Korea), and after collecting the virus, the Top TM Vrial DNA/RNA Exraction Kit (Sissun Biomaterials, Korea) is used. isolates the DNA and RNA of the virus, and detects SARS-CoV-2 using the AQ-TOP TM COVID-19 Rapid Kit (Sisun Biomaterials, Korea), s, Korea) and PCR amplification takes about 15 minutes (Table 2).
  • the "virus isolation step” proceeds with an automatic magnetic bead system, and it is preferable that it takes about 20 minutes to proceed with the 48 test. Alternatively, it proceeds with the Column/Syringe System. and preferably 10 minutes per 24 tests or 10 minutes per 10 tests.
  • the "diagnosis/analysis step" of the emergency test is PCR amplification by applying AQ-TOPTM COVID-19 Rapid Kit (SEASUN) and SEASUN Bio Isothermal Amplification Equipment (Seasun Biomaterials, Korea), Preferably, it takes about 15 minutes.
  • the departure inspection and the emergency inspection may be characterized by performing isothermal amplification PCR using the AQ-TOPTM COVID-19 Rapid Kit.
  • the emergency test be performed on a positive discriminant in a pooling test.
  • the sample collection may be characterized in that it is collected through the oral cavity (Oral) or the nasal cavity (Nasal).
  • a sample with a body temperature of 37.5° C. or lower is collected through the oral cavity, and a sample with a body temperature of 37.5° C. or higher and suspected symptoms is collected through the nasal cavity.
  • the AQ-TOP TM COVID-19 Rapid Kit may be characterized by including a primer pair for SARS-CoV-2 gene amplification selected from the following (i) to (xi):
  • (x) a primer pair consisting of a forward primer of SEQ ID NO: 19 and a reverse primer of SEQ ID NO: 20;
  • (xi) a primer pair consisting of a forward primer of SEQ ID NO: 21 and a reverse primer of SEQ ID NO: 22.
  • the AQ-TOP TM COVID-19 Rapid Kit may include a PNA probe for diagnosis of SARS-CoV-2 represented by one or more sequences selected from the group consisting of SEQ ID NOs: 23-30. .
  • the quarantine system of the present invention is a quarantine system, characterized in that it is performed at an airport, a port, or a large-scale facility.
  • the primer pair included in the AQ-TOPTM COVID-19 Rapid Kit is a primer pair capable of amplifying the ORF1ab gene and N gene of SARS-CoV-2 virus, and amplified by the primer pair The amplified product is detected by the PNA probe of SEQ ID NOs: 33 to 48.
  • the PNA probe is not limited, but may be characterized in that a reporter or a quencher is bound.
  • the probe of the present invention may bind to both ends of a reporter and a fluorescent material of a quencher capable of quenching reporter fluorescence, and may include an intercalating fluorescent material.
  • the reporter may be one or more selected from the group consisting of FAM (6-carboxyfluorescein), HEX, Texas red, JOE, TAMRA, CY5, CY3, Alexa680, and the quencher is TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, It is preferred, but not limited to, BHQ2 or Dabcyl.
  • the intercalating fluorescent material is acridine homodimer and its derivatives, acridine orange and its derivatives, 7-aminoactinomycin D (7-AAD) and derivatives thereof, Actinomycin D and derivatives thereof, ACMA (9-amino-6-chloro-2-methoxyacridine) and derivatives thereof, DAPI and derivatives thereof, dihydroethidium (Dihydroethidium) and its derivatives, ethidium bromide and its derivatives, ethidium homodimer-1 (EthD-1) and its derivatives, ethidium homodimer-2 (EthD-2) and its derivatives, ethidium Ethidium monoazide and its derivatives, Hexidium iodide and its derivatives, bisbenzimide (Hoechst 33258) and its derivatives, Hoechst 33342 and its derivatives, ho Hoechst 34580 and its derivatives, hydroxystilbamidine and its derivatives, LDS
  • the AQ-TOPTM COVID-19 Rapid Kit is a control material; a primer pair of a forward primer of SEQ ID NO: 31 and a reverse primer of SEQ ID NO: 32, a primer pair of a forward primer of SEQ ID NO: 33 and a reverse primer of SEQ ID NO: 34 for detecting this; And it may be characterized in that it further comprises a PNA probe of SEQ ID NOs: 35-37, but is not limited thereto.
  • the internal control material can be used without limitation as long as it is a gene that can confirm the success or failure of the polymerase chain reaction in a clinical specimen regardless of the presence or absence of SARS-CoV-2, but preferably a homosapiens species Rnase P gene It may be characterized as an RNA region of
  • the diagnosis/analysis step is preferably performed as the following step.
  • the specimen sample is a DNA or RNA molecule, and the molecule may be in a double-stranded or single-stranded form.
  • the nucleic acid as an initial material is single-stranded RNA
  • cDNA complementary DNA
  • the nucleic acid as a starting material is double-stranded, it is preferable to make the two strands into a single-stranded or partially single-stranded form.
  • Methods known to separate strands include, but are not limited to, heat, alkali, formamide, urea and glycoxal treatment, enzymatic methods (eg, helicase action) and binding proteins.
  • strand separation can be achieved by heat treatment at a temperature of 80°C to 105°C.
  • the gene is not limited, but may be the ORF1ab gene or N gene of SARS-CoV-2.
  • the analysis method is not limited, but it may be characterized in that it is performed by PCR, reverse transcription real time PCR, or isothermal amplification PCR method.
  • step b) may be characterized in that it further comprises a control material.
  • the internal control material can be used without limitation as long as it is a gene that can confirm the success of the reverse transcription polymerase chain reaction in a clinical sample regardless of the presence or absence of the novel coronavirus (SARS-CoV-2), but preferably It can be characterized as an RNA region of the homosapiens species RNase P gene.
  • SARS-CoV-2 novel coronavirus
  • the 'target nucleic acid', 'synthetic DNA' or 'artificial oligo' of the present invention means a nucleic acid sequence to be detected, and the nucleic acid sequence of a 'target gene' encoding a protein having physiological and biochemical functions. It contains a specific site and is annealed or hybridized with a primer or probe under hybridization, annealing or amplification conditions.
  • Hybridization' in the present invention means that complementary single-stranded nucleic acids form double-stranded nucleic acids. Hybridization may occur when complementarity between two nucleic acid strands is perfect (perfect match) or even when some mismatched bases are present. The degree of complementarity required for hybridization may vary depending on hybridization conditions, and in particular may be controlled by temperature.
  • the PNA probe containing the reporter and quencher of the present invention generates a fluorescent signal after hybridization with the target nucleic acid, and the presence or absence of the target nucleic acid can be detected through analysis.
  • Example 1 Preparation of primers and detection probes for SARS-CoV-2 gene amplification
  • primers of SEQ ID NOs: 1 to 18 were prepared as shown in Table 3 below.
  • PNA probes of SEQ ID NOs: 23 to 30 were prepared, and fluorescence and quenchers were respectively coupled to both ends of the probe sequence for multiple amplification (Table 4).
  • a primer capable of detecting this was prepared using the RNA 150bp region of the Rnase P gene of Homosapiens as a target nucleic acid (Table 5), and the amplification product of the control material was prepared.
  • a detectable PNA probe was prepared (Table 6).
  • the 4S Seasun Smart Shield System
  • the AQ-TOP TM COVID-19 Plus kit it is possible to quickly and accurately determine whether the person is infected with COVID-19, so that the population moves frequently in airports, ports, etc. It is effective for rapid quarantine, large-scale gatherings, and total inspection in areas with mass infection.

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Abstract

The present invention relates to a COVID-19 quarantine system using isothermal amplification-based molecular diagnostics for detecting SARS-CoV-2, and in particular, to a quarantine system in an airport, a port, a massive rally, or a superspreader area. According to the present invention, it is possible to quickly and accurately test COVID-19 infection on a large scale, and thus the present invention is effective for the quarantining of mass service facilities, such as airports, ports, and gymnasiums.

Description

등온증폭 기반의 분자진단법을 이용한 고위험성 전염병에 대한 검역시스템Quarantine system for high-risk infectious diseases using molecular diagnostics based on isothermal amplification
본 발명은 고위험성 전염병인 코로나19 바이러스 감염병에 대한 방역 및 검역시스템에 관한 것으로, 더욱 자세하게는 SARS-CoV-2 검출용 등온증폭(isothermal amplification) 기반의 분자 진단법(molecular diagnostics)을 이용한 코로나 19 검역시스템, 특히 공항, 항만, 대규모 집회, 집단감염 지역에서의 검역시스템에 관한 것이다.The present invention relates to a quarantine and quarantine system for a high-risk infectious disease, Corona 19 virus, and more specifically, Corona 19 quarantine using isothermal amplification-based molecular diagnostics for SARS-CoV-2 detection It relates to the system, especially the quarantine system in airports, ports, large gatherings, and cluster infection areas.
코로나바이러스는 동물들에서 위장병 및 호흡기질환과 같은 다양한 질환을 일으킬 수 있다고 알려져 있다. 사람에게 감염되는 사람 코로나바이러스는 1960대 발견된 HCoV-229E와 HCoV-OC43, 사스 대유행 이후 발견된 HCoV-NL63(2004년)과 HCoV-HKU1(2005년)로 이들은 일반적으로 상기도 감염증과 관계가 있다고 알려져 있으나 면역결핍환자들에게는 심각한 폐질환을 유도하기도 한다. 주로 겨울이나 이른 봄에 코로나바이러스에 대한 감염률이 증가된다고 보고되고 있고, 성인 감기환자 중 코로나바이러스가 원인 병원체인 비율이 상당히 높다고 알려져 있다. 2003년 중증급성호흡기증후군(Severe Acute Respiratory Syndrome, SARS)을 일으키는 사스 코로나바이러스(SARS-CoV)가 처음 발견되었으며 세계보건기구(WHO) 보고에 따르면 2002년에서 2003년 동안 전 세계적으로 8,273명의 환자와 775명의 사망자(치사율 약 10%)가 발생하였고, 2004년까지 추가적인 환자 발생과 사망자가 보고되었다(Saif LJ., Rev. Sci. Tech., 23:643, 2004).Coronaviruses are known to cause a variety of diseases in animals, such as gastrointestinal and respiratory diseases. Human coronaviruses that infect humans are HCoV-229E and HCoV-OC43 discovered in the 1960s, and HCoV-NL63 (2004) and HCoV-HKU1 (2005) discovered after the SARS pandemic, which are generally not associated with upper respiratory tract infections. It is known that it can lead to serious lung disease in immunodeficiency patients. It is reported that the infection rate for coronavirus increases mainly in winter or early spring, and it is known that the proportion of the pathogen that is the cause of the coronavirus among adult cold patients is quite high. SARS-CoV, which causes Severe Acute Respiratory Syndrome (SARS), was first identified in 2003, and according to the World Health Organization (WHO) report, 8,273 patients and There were 775 deaths (mortality rate of about 10%), with additional cases and deaths reported by 2004 (Saif LJ., Rev. Sci. Tech ., 23:643, 2004).
2012년 9월 사스와 유사한 고열, 기침, 호흡곤란 등의 호흡기 증상을 나타내는 중중 호흡기질환 환자가 발생하였고, 이 원인 병원체는 기존의 알려진 바이러스와는 다른 신종 코로나바이러스(HCoV-EMC)로 밝혀졌다. 이 바이러스는 중동호흡기 증후군 코로나바이러스 또는 메르스 코로나바이러스(Middle East Respiratory Syndrome Coronavirus; MERS-CoV)로 불리며, 사람에게 감염되었을 때에 중증의 호흡기 질환을 유발하여 사망에 이르게 하며(Zaki AM et al., N Engl J Med, 367:1814, 2012), 2015년 5월 우리나라에서 발생하여 38명의 사망자를 낸 바 있다. In September 2012, a patient with severe respiratory disease showing respiratory symptoms such as high fever, cough, and shortness of breath similar to SARS occurred, and the pathogen was found to be a novel coronavirus (HCoV-EMC) different from the previously known virus. This virus, called Middle East Respiratory Syndrome Coronavirus (MERS-CoV), causes severe respiratory illness and death when infected in humans (Zaki AM et al., N Engl J Med , 367:1814, 2012), occurred in Korea in May 2015, causing 38 deaths.
코로나바이러스감염증-19는 2019년 12월 중국 우한시에서 발생한 바이러스성 호흡기 질환이다. '우한 폐렴', '신종코로나바이러스감염증', '코로나19'라고도 한다. 신종 코로나바이러스에 의한 유행성 질환으로 호흡기를 통해 감염되며, 증상이 거의 없는 감염 초기에 전염성이 강한 특징을 보인다. 감염 후에는 인후통, 고열, 기침, 호흡곤란 등의 증상을 거쳐 폐렴으로 발전한다. 2020년 3월 전세계로 확산되자, 세계보건기구는 이 질환에 대해 팬데믹을 선언했다.Coronavirus Infectious Disease-19 is a viral respiratory disease that occurred in Wuhan, China in December 2019. It is also called 'Wuhan pneumonia', 'novel coronavirus infection', 'corona 19'. It is an epidemic disease caused by the novel coronavirus, which is transmitted through the respiratory tract, and shows strong contagious characteristics in the early stages of infection with few symptoms. After infection, symptoms such as sore throat, high fever, cough, and shortness of breath develop into pneumonia. After it spread worldwide in March 2020, the World Health Organization declared the disease a pandemic.
코로나바이러스감염증-19(Covid-19)는 주로 호흡기로 전염된다. 감염되었을 경우 바이러스는 폐를 침범하며, 고열과 기침, 호흡곤란 등의 증상이 발생하고 폐렴과 유사한 증상을 보인 끝에 심한 경우 폐포가 손상되어 호흡 부전으로 사망에 이르기도 한다. 잠복기는 3~7일이지만 최장 14일까지 이어지기도 한다. 2020년 1월 30일 중국에서는 잠복기가 23일까지 늘어난 사례가 있다고 발표했다. 코로나19는 증상이 나타나지 않는 잠복기 중에도 전염되는 사례가 있다고 보고되었다.Coronavirus Infectious Disease-19 (Covid-19) is mainly transmitted through the respiratory tract. When infected, the virus invades the lungs, causing symptoms such as high fever, coughing, and shortness of breath, and after showing symptoms similar to pneumonia, in severe cases, the alveoli are damaged, leading to death from respiratory failure. The incubation period is 3 to 7 days, but can last up to 14 days. On January 30, 2020, China announced that there were cases where the incubation period was extended to 23 days. It has been reported that COVID-19 is transmitted even during the incubation period when symptoms do not appear.
코로나바이러스감염증-19의 잠복기 무증상 시에도 전염되는 특성과 강한 전염성으로 인해 인접한 지역으로 신속하게 확산되기 때문에 조기 검출에 의한 환자의 격리 및 관리가 매우 중요하다.The isolation and management of patients by early detection is very important because COVID-19 spreads rapidly to neighboring areas due to its contagious nature and strong contagiousness even in the incubation period of COVID-19.
현재 신종 코로나바이러스 감염증(코로나19)을 일으키는 SARS-CoV-2는 기본적으로 '판코로나바이러스 검사법(Conventional PCR)'과 염기서열 분석으로 진단한다. 판코로나바이러스 검사법은 신종코로나바이러스를 포함한 모든 코로나바이러스의 존재 유무를 우선 검사하는 것으로, 음성으로 판정되면 코로나바이러스 감염이 아님을 의미한다. 만일 양성인 경우에는 감기를 일으키는 다른 코로나바이러스인지, 신종코로나바이러스인지 유무를 유전자염기서열 분석을 통해 판단한다. 이에 따라 진단에 1~2일 정도 소요된다.SARS-CoV-2, which currently causes novel coronavirus infection (COVID-19), is basically diagnosed by 'conventional PCR' and sequencing analysis. The pan-coronavirus test method first tests for the presence or absence of all coronaviruses, including the novel coronavirus, and if it is negative, it means that it is not a coronavirus infection. If it is positive, it is determined whether it is another coronavirus causing a cold or a novel coronavirus through gene sequencing. Therefore, it takes 1 to 2 days for diagnosis.
한편, 한국의 질병관리본부는 해외로부터의 코로나-19 감염자의 입국을 차단하기 위하여, 국내로 입국하는 내·외국인을 대상으로 특별검역을 실시하고 있으며, 아시아 5개국, 유럽 6개국 등 총 11개국 출발 항공노선에 적용되던 특별입국 절차를 2020년 3월 19일부터 모든 국가에서 출발한 항공노선으로 확대하여 적용하고 있다. 특별입국절차는 입국 전 항공기 내에서 건강상태질문서, 특별검역신고서 등을 작성하고, 입국장 검역에서 발열체크, 건강상태질문서 제출 및 유증상 여부를 확인하고 있으며, 증상이 있을 시 검역소 내 격리시설에서 격리해 검체채취와 진단검사를 실시하고 있다. 아울러 '모바일 자가진단앱'을 설치하여 입국 이후 14일간 발열, 기침 등 건강상태를 모니터링하고 있다. Meanwhile, the Korea Centers for Disease Control and Prevention (KCDC) is carrying out special quarantine for domestic and foreign nationals entering Korea in order to block the entry of people infected with COVID-19 from overseas. From March 19, 2020, the special immigration procedure applied to departure air routes has been expanded and applied to air routes departing from all countries. The special entry procedure involves filling out a health status questionnaire and special quarantine report on board the aircraft before entering the country, temperature check at the arrival hall quarantine, submitting a health status questionnaire, and checking for symptoms. quarantined, collecting samples and conducting diagnostic tests. In addition, a 'mobile self-diagnosis app' is installed to monitor health conditions such as fever and cough for 14 days after entry.
질병관리본부는 2020년 1월 31일부터는 검사속도와 편의성이 향상된 '실시간 유전자 증폭검사(Real Time RT-PCR)'를 통해 진단하고 있는데 이 검사방법으로는 결과 확인을 위해 6시간 정도가 소요된다(도 1).From January 31, 2020, the Korea Centers for Disease Control and Prevention is diagnosing through 'Real Time RT-PCR', which has improved test speed and convenience, and this test method takes about 6 hours to confirm the result. (Fig. 1).
현재 민감도 및 특이도가 보다 향상된 효과적인 검출이 가능한 검출 방법이 필요한 실정이며, 민감도와 특이도가 향상된 검출방법을 이용한 신속하고 정확한 공항검역 시스템의 개발이 요구되고 있다. Currently, there is a need for a detection method capable of effective detection with improved sensitivity and specificity, and the development of a rapid and accurate airport quarantine system using a detection method with improved sensitivity and specificity is required.
이에, 발명자들은 해외입출국자들에 대한 코비드-19 검역을 보다 신속하고 정확하게 수행하는 방법을 개발하고자 예의 노력한 결과, SARS-CoV-2의 ORF1ab 유전자 및 N 유전자를 특이적으로 증폭하는 프라이머와 이를 검출하는 PNA 프로브를 포함하는 AQ-TOP COVID-19 Rapid 키트를 이용하여, 유증상자의 검체를 검사하는 경우, 기존의 방법보다 보다 신속하고 빠르게 공항검역을 진행할 수 있다는 것을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the inventors made diligent efforts to develop a method for more rapidly and accurately performing Covid-19 quarantine for overseas arrivals and departures. As a result, a primer that specifically amplifies the ORF1ab gene and N gene of SARS-CoV-2 and the When using the AQ-TOP COVID-19 Rapid kit containing a PNA probe to detect, when examining a sample from a symptomatic person, it was confirmed that airport quarantine can be performed more quickly and quickly than the existing method, and completed the present invention did it
발명의 요약Summary of the invention
본 발명의 목적은 코로나-19 바이러스 감염자 또는 감염 의심자를 신속하게 선별하기 위한 AQ-TOPTM COVID-19 Rapid 키트를 이용한 검역시스템, 구체적으로는 4S(Seasun Smart Shield System)라 명명된 새로운 공항, 항만, 대규모 집회, 집단감염 지역 등에 대한 검역시스템을 제공하는데 있다. An object of the present invention is a quarantine system using the AQ-TOP TM COVID-19 Rapid kit to quickly screen a person infected with or suspected of being infected with the COVID-19 virus, specifically a new airport, port named 4S (Seasun Smart Shield System) , to provide a quarantine system for large gatherings and cluster infection areas.
상기 목적을 달성하기 위하여, 본 발명은 SARS-CoV-2 검출용 AQ-TOPTM COVID-19 Rapid 키트를 이용한 코로나 19 검역시스템, 특히 공항, 항만, 대규모 집회, 집단감염 지역 등에 대한 검역시스템을 제공한다. In order to achieve the above object, the present invention provides a COVID-19 quarantine system using AQ-TOP TM COVID-19 Rapid kit for detecting SARS-CoV-2, in particular, a quarantine system for airports, ports, large gatherings, mass infection areas, etc. do.
도 1은 기존 COVID-19 검사제품의 현황을 도식화하여 나타낸 것이다.1 is a schematic representation of the current state of the existing COVID-19 test product.
도 2는 본 발명에 따른 "시선 스마트 쉴드 시스템(Seasun Smart Shield System)"을 이용한 검역 시스템을 나타낸 것이다. 2 shows a quarantine system using the "Seasun Smart Shield System" according to the present invention.
도 3은 본 발명에 따른 검역시스템에서 풀링테스트 방법을 나타낸 것이다.3 shows a pulling test method in the quarantine system according to the present invention.
도 4는 본 발명에 따른 검역시스템에서 단일테스트 방법을 나타낸 것이다.4 shows a single test method in the quarantine system according to the present invention.
도 5는 본 발명에 따른 검역시스템에서 긴급테스트 방법을 나타낸 것이다.5 shows an emergency test method in the quarantine system according to the present invention.
발명의 상세한 설명 및 바람직한 구현예DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
본 발명에서는 기존의 방법보다 보다 신속하고 빠르게 대규모 검역을 진행할 수 있는 검역시스템을 개발하였으며, SARS-CoV-2 바이러스의 ORF1ab 유전자 및 N 유전자를 증폭할 수 있는 프라이머 쌍 및 상기 프라이머 쌍에 의하여 증폭된 산물을 높은 민감도와 특이도로 검출할 수 있는 PNA 프로브를 사용하는 유전자 증폭방법으로, 공항 검역소, 항만, 실내 집단시설, 집단 감염지역 등 다수의 인원을 단시간에 검사해야하는 경우, 기존 진단방법보다 신속하고 정확하게 SARS-CoV-2 감염여부를 진단할 수 있다는 것을 확인하고, "시선 스마트 쉴드 시스템(Seasun Smart Shield System, 4S)"을 적용한 검역시스템을 개발하였다.In the present invention, a quarantine system that can perform large-scale quarantine faster and faster than the existing method has been developed, and a primer pair capable of amplifying the ORF1ab gene and N gene of SARS-CoV-2 virus, and the primer pair amplified by the This is a gene amplification method that uses a PNA probe that can detect products with high sensitivity and specificity. When a large number of people such as airport quarantine stations, ports, indoor collective facilities, and collective infection areas need to be tested in a short time, it is faster and faster than existing diagnostic methods. After confirming that it can accurately diagnose SARS-CoV-2 infection, a quarantine system applied with the "Seasun Smart Shield System (4S)" was developed.
따라서, 본 발명은 SARS-CoV-2 검출용 AQ-TOP™ COVID-19 Rapid Kit를 이용한 코로나19 검역시스템에 관한 것이다.Accordingly, the present invention relates to a COVID-19 quarantine system using AQ-TOP™ COVID-19 Rapid Kit for SARS-CoV-2 detection.
본 발명은 유전자 증폭을 통한 분자진단방법을 이용한 코로나19 검역시스템이며, 특히, 등온증폭기반 분자진단방법을 사용하는 것이 바람직하나 이에 한정하는 것은 아니다. The present invention is a COVID-19 quarantine system using a molecular diagnostic method through gene amplification, and in particular, it is preferable to use an isothermal amplification-based molecular diagnostic method, but is not limited thereto.
본 발명에서, 일 예로, 공항 검역시스템은 입국 검사, 출국 검사 및 긴급 검사로 구성되며, 입국검사는 해외 방문자 또는 외국인의 입국 시, 국내 감염확산을 사전에 차단/격리하여 코로나19를 예방하기 위한 검사로, 빠른 시간 내에 많은 검사를 수행하여야 한다.In the present invention, as an example, the airport quarantine system consists of entry inspection, departure inspection, and emergency inspection. As an inspection, many inspections must be performed in a short time.
본 발명에 있어서, 상기 풀링테스트(pooling test), 단일테스트(single test) 및 긴급 테스트(emergency test)는 모두 신원확인 단계; 검체 채취 단계; 바이러스 분리 단계; 및 진단/분석 단계로 진행되는 것을 특징으로 할 수 있다(도 2). In the present invention, the pooling test (pooling test), the single test (single test) and the emergency test (emergency test) are all identification steps; sampling step; virus isolation step; And it may be characterized in that it proceeds to the diagnosis / analysis step (FIG. 2).
본 발명에 있어서, 상기 풀링테스트(pooling test), 단일테스트(single test) 및 긴급 테스트(emergency test)는 모두 신원확인 단계가 필요하며, 모두 임상검사 진단 동의서 및 개별인식표가 필요하며, 상기 임상검사 진단 동의서는 일 예로, 공항에서 입국하는 경우는 기내에서 동의서를 배부 후 수거하는 것이 바람직하며, 출국검사의 경우는 전용 대기실 내에서 배부 및 수거를 진행하는 것이 바람직하다. 상기 개별인식표는 팔찌 또는 신분증(여권, 주민등록증, 운전면허증 등)에 스티커를 부착하는 방식으로 수행할 수 있다(도 3). In the present invention, the pooling test, the single test, and the emergency test all require an identification step, and all require a clinical examination diagnosis consent form and an individual identification tag, and the clinical examination For example, in the case of entering from the airport, it is desirable to collect the consent form after distributing it on board, and in the case of departure inspection, it is desirable to distribute and collect it in the dedicated waiting room. The individual identification tag may be performed by attaching a sticker to a bracelet or identification card (passport, resident registration card, driver's license, etc.) (FIG. 3).
본 발명에서는 풀링테스트(pooling test)는 3~4명의 검체를 하나의 튜브에 넣어 검사하는 방식으로 진행하는 것을 특징으로 한다.In the present invention, the pooling test is characterized in that it is carried out in a manner in which three or four samples are put into one tube and tested.
본 발명에서, 풀링테스트(pooling test)는 3~4명의 검체를 1개의 VTM 튜브에 넣어 1그룹으로 지정하여 검사하는 Big Screening system으로 진행하며, 빠른 시간 안에 대규모 검사를 수행하는 것이 바람직하며, 최대 760명을 1시간에 검사할 수 있으며, 감염 확인 시에는 추가 정밀검사가 필요하다. In the present invention, the pooling test is performed with a Big Screening system in which 3 to 4 specimens are placed in one VTM tube and designated as one group for inspection. It can test 760 people in an hour, and additional detailed tests are required to confirm the infection.
본 발명의 일양태에서 Big Screening system은 TopTM Virus Collection kit(시선바이오머티리얼스, 한국)를 사용하여, 바이러스를 수집한 후, TopTM Vrial DNA/RNA Exraction Kit(시선바이오머티리얼스, 한국)를 사용하여, 바이러스의 DNA 및 RNA를 분리하고, AQ-TOPTM CIVID-19 Rapid Kit(시선바이오머티리얼스, 한국)를 사용하여 SARS-CoV-2를 검출하였으며, 유전자 증폭은 CFX96(Bio-Rad) 또는 ABI7500(ABI) 장비를 사용하여 증폭을 수행한다(표 1). In one aspect of the present invention, the Big Screening system uses the Top TM Virus Collection kit (Sisun Biomaterials, Korea), and after collecting the virus, the Top TM Vrial DNA/RNA Exraction Kit (Sissun Biomaterials, Korea) is used. was used to isolate virus DNA and RNA, and SARS-CoV-2 was detected using AQ-TOP TM CIVID-19 Rapid Kit (Sisun Biomaterials, Korea), and gene amplification was performed by CFX96 (Bio-Rad). Alternatively, amplification is performed using an ABI7500 (ABI) instrument (Table 1).
본 발명의 일 실시예에 있어서, 상기 공항에서의 입국 시 풀링테스트(pooling test)의 검체 체취는 발열기준 (37.5℃)에 따라 37.5℃이하는 “구강용 면봉(Oral swab)”, 37.5℃ 이상은 “비강용 면봉(Nasal swab)”으로 샘플링을 진행하며, 전문 의료인 및 기내 승무원이 동행하여, Connection Bridge에서 검체를 채취하며, 기내 좌석별로 1열-1그룹화 (4명/tube)하여 검체 채취를 진행하는 것이 바람직하다. In one embodiment of the present invention, the sample odor of the pooling test upon entry at the airport is 37.5 ° C or less according to the fever standard (37.5 ° C), "Oral swab", 37.5 ° C or more Sampling is carried out with a “Nasal swab”, and a medical professional and flight attendant accompany them to collect a sample at the Connection Bridge, and collect samples by grouping 1 row-1 for each seat in the cabin (4 people/tube) It is preferable to proceed with
본 발명에 있어서, 상기 "바이러스 분리 단계"는 자동 자석비드 시스템(Automation Magnetic bead System)으로 진행하며, 48 테스트를 진행하는데 약 20분이 소요되는 것이 바람직하다.In the present invention, the "virus isolation step" proceeds with an automatic magnetic bead system (Automation Magnetic bead System), and it is preferable that it takes about 20 minutes to proceed with the 48 test.
본 발명에 있어서, 상기 풀링테스트(pooling test)의 "진단/분석단계"는 AQ-TOP™ COVID-19 Rapid Kit (시선바이오머티리얼스, 한국)와 CFX96 혹은 CFX384장비 (Bio-Rad 또는 ABI)를 적용하여 PCR 증폭 진행. 약 30분 소요되는 것이 바람직하다.In the present invention, the "diagnosis/analysis step" of the pooling test is performed using AQ-TOP™ COVID-19 Rapid Kit (Sightsun Biomaterials, Korea) and CFX96 or CFX384 equipment (Bio-Rad or ABI). Apply to proceed with PCR amplification. It is preferable that it takes about 30 minutes.
Figure PCTKR2021008506-appb-img-000001
Figure PCTKR2021008506-appb-img-000001
본 발명에서는 단일테스트(single test)는 단일 테스트로 1명의 검체를 하나의 튜브에 넣어 검사를 진행하는 것을 특징으로 한다(도 4).In the present invention, the single test is characterized in that the test is performed by putting one specimen in one tube as a single test (FIG. 4).
본 발명에서, 단일테스트(single test)는 신속하고 정확한 검사를 통하여 빠른 결과 보고와 상황 종료를 원활하게 진행할 수 있도록 한다. In the present invention, a single test enables a quick result report and a smooth end of a situation through a quick and accurate test.
본 발명에서, 상기 단일테스트(single test)는 1명의 검체를 1개의 VTM 튜브에 넣어 1:1로 진행되는 단일 검사 시스템을 적용하는 Urgent Screening system으로 진행하며, 빠른 시간 안에 정확한 진단이 가능하며, 30분 내에 최대 64명의 검사가 가능하다. In the present invention, the single test is carried out with the Urgent Screening system in which a single test system is applied 1:1 by putting one specimen into one VTM tube, and accurate diagnosis is possible in a short time, Up to 64 people can be tested in 30 minutes.
본 발명의 일양태에서 Urgent Screening system은 TopTM Virus Collection kit(시선바이오머티리얼스, 한국)를 사용하여, 바이러스를 수집한 후, TopTM Vrial DNA/RNA Exraction Kit(시선바이오머티리얼스, 한국)를 사용하여, 바이러스의 DNA 및 RNA를 분리하고, AQ-TOPTM COVID-19 Rapid Kit(시선바이오머티리얼스, 한국)를 사용하여 SARS-CoV-2를 검출하며, 시선바이오 등온증폭장비(시선바이오머티리얼스, 한국)를 적용하여 PCR 증폭을 진행하며 약 15분이 소요된다(표 2). In one aspect of the present invention, the Urgent Screening system uses the Top TM Virus Collection kit (Siksun Biomaterials, Korea), and after collecting the virus, the Top TM Vrial DNA/RNA Exraction Kit (Sissun Biomaterials, Korea) is used. isolates the DNA and RNA of the virus, and detects SARS-CoV-2 using the AQ-TOP TM COVID-19 Rapid Kit (Sisun Biomaterials, Korea), s, Korea) and PCR amplification takes about 15 minutes (Table 2).
본 발명에 있어서, 상기 단일테스트(single test) 시 발열기준 (37.5℃)에 따라 37.5℃이하는 단일테스트(single test)를 패스하며, 37.5℃ 이상은 “비강용 면봉(Nasal swab)”으로 샘플링을 진행하며, 전문 의료인 및 전용 대기실 내에서 검체를 채취한다. In the present invention, according to the fever standard (37.5℃) in the single test, 37.5℃ or less passes the single test, and 37.5℃ or more is sampled with a “Nasal swab” and collect samples in the waiting room with specialized medical personnel.
본 발명에 있어서, 상기 "바이러스 분리 단계"는 자동 자석비드 시스템(Automation Magnetic bead System)으로 진행하며, 48 테스트를 진행하는데 약 20분이 소요되는 것이 바람직하다.In the present invention, the "virus isolation step" proceeds with an automatic magnetic bead system (Automation Magnetic bead System), and it is preferable that it takes about 20 minutes to proceed with the 48 test.
본 발명에 있어서, 상기 단일테스트(single test)의 "진단/분석 단계"는 AQ-TOP™ COVID-19 Rapid Kit (SEASUN)와 시선바이오 등온증폭 장비(시선바이오머티리얼스, 한국)를 적용하여 PCR 증폭을 진행하며 약 15분이 소요되는 것이 바람직하다.In the present invention, the "diagnosis/analysis step" of the single test is PCR by applying AQ-TOP™ COVID-19 Rapid Kit (SEASUN) and Sesun Bio isothermal amplification equipment (Seasun Biomaterials, Korea) It is preferable that the amplification process takes about 15 minutes.
Figure PCTKR2021008506-appb-img-000002
Figure PCTKR2021008506-appb-img-000002
본 발명에서 긴급테스트(emergency test)는 단일 테스트로 1명의 검체를 하나의 튜브에 넣어 검사를 진행하는 것을 특징으로 한다(도 5).In the present invention, the emergency test is characterized in that the test is performed by putting one specimen in one tube as a single test (FIG. 5).
본 발명에서, 긴급테스트(emergency test)는 실내 시설 및 지역에서의 감염 확산을 사전 차단/격리하여 신속하고 정확한 검역시스템으로 감염병 검사를원활하게 진행할 수 있도록 하며, 발열과 기저질환 보유한 코로나19 의심자를 대상을 1차적으로 검사하며, Pooling test 통해 감염병 양성 판정 나온 그룹 등을 대상으로 한다. In the present invention, the emergency test prevents/isolates the spread of infection in indoor facilities and areas in advance so that the examination for infectious diseases can be carried out smoothly with a rapid and accurate quarantine system, Subjects are tested primarily, and groups that are positive for infectious diseases through pooling test are targeted.
본 발명에서, 상기 긴급테스트는 1명의 검체를 1개의 VTM 튜브에 넣어 1:1로 진행되는 단일 검사 시스템을 적용하는 긴급선별법(Urgent Screening system)으로 진행하며, 빠른 시간 안에 정확한 진단이 가능하며, 30분 내에 최대 64명의 검사가 가능하다. In the present invention, the urgent test is carried out by an urgent screening system that applies a single test system that is conducted 1:1 by putting one specimen into one VTM tube, and accurate diagnosis is possible in a short time, Up to 64 people can be tested in 30 minutes.
본 발명의 일양태에서 Urgent Screening system은 TopTM Virus Collection kit(시선바이오머티리얼스, 한국)를 사용하여, 바이러스를 수집한 후, TopTM Vrial DNA/RNA Exraction Kit(시선바이오머티리얼스, 한국)를 사용하여, 바이러스의 DNA 및 RNA를 분리하고, AQ-TOPTM COVID-19 Rapid Kit(시선바이오머티리얼스, 한국)를 사용하여 SARS-CoV-2를 검출하며, 시선바이오 등온증폭장비(시선바이오머티리얼스, 한국)를 적용하여 PCR 증폭을 진행하며 약 15분이 소요된다(표 2). In one aspect of the present invention, the Urgent Screening system uses the Top TM Virus Collection kit (Siksun Biomaterials, Korea), and after collecting the virus, the Top TM Vrial DNA/RNA Exraction Kit (Sissun Biomaterials, Korea) is used. isolates the DNA and RNA of the virus, and detects SARS-CoV-2 using the AQ-TOP TM COVID-19 Rapid Kit (Sisun Biomaterials, Korea), s, Korea) and PCR amplification takes about 15 minutes (Table 2).
본 발명에 있어서, 상기 긴급테스트시 발열기준 (37.5℃)에 따라 37.5℃이하는 출국검사를 통과하며, 37.5℃ 이상은 “비강용 면봉(Nasal swab)”으로 샘플링을 진행하며, 전문 의료인이 전용 대기실 내에서 검체를 채취한다. In the present invention, according to the fever standard (37.5℃) during the emergency test, 37.5℃ or less passes the departure inspection, and 37.5℃ or more, sampling is carried out with a “Nasal swab”, and professional medical personnel only Specimens are collected in the waiting room.
본 발명에 있어서, 상기 "바이러스 분리 단계"는 자동 자석비드 시스템(Automation Magnetic bead System)으로 진행하며, 48 테스트를 진행하는데 약 20분이 소요되는 것이 바람직하며, 다른 방법으로, Column/Syringe System으로 진행하며, 24 테스트 당 10 분 또는 10 테스트 당 10분이 소요되는 것이 바람직하다. In the present invention, the "virus isolation step" proceeds with an automatic magnetic bead system, and it is preferable that it takes about 20 minutes to proceed with the 48 test. Alternatively, it proceeds with the Column/Syringe System. and preferably 10 minutes per 24 tests or 10 minutes per 10 tests.
본 발명에 있어서, 상기 긴급검사의 "진단/분석 단계"는 AQ-TOP™ COVID-19 Rapid Kit (SEASUN)와 시선바이오 등온증폭 장비(시선바이오머티리얼스, 한국)를 적용하여 PCR 증폭을 진행하며 약 15분이 소요되는 것이 바람직하다.In the present invention, the "diagnosis/analysis step" of the emergency test is PCR amplification by applying AQ-TOP™ COVID-19 Rapid Kit (SEASUN) and SEASUN Bio Isothermal Amplification Equipment (Seasun Biomaterials, Korea), Preferably, it takes about 15 minutes.
본 발명에 있어서, 상기 출국검사 및 상기 긴급검사는 AQ-TOP™ COVID-19 Rapid Kit를 이용하여 등온증폭(Isothermal Amplification) PCR로 수행하는 것을 특징으로 할 수 있다. In the present invention, the departure inspection and the emergency inspection may be characterized by performing isothermal amplification PCR using the AQ-TOP™ COVID-19 Rapid Kit.
본 발명에 있어서, 상기 긴급테스트는 풀링테스트에서 양성판별자에 대하여 시행하는 것이 바람직하다. In the present invention, it is preferable that the emergency test be performed on a positive discriminant in a pooling test.
본 발명에 있어서, 상기 검체 채취는 구강(Oral) 또는 비강(Nasal)을 통하여 채취하는 것을 특징으로 할 수 있다. In the present invention, the sample collection may be characterized in that it is collected through the oral cavity (Oral) or the nasal cavity (Nasal).
본 발명에 있어서, 상기 풀링테스트의 경우, 체온이 37.5℃ 이하의 유증상자는 구강(Oral)을 통하여 검체를 채취하고, 37.5℃ 이상 및 의심증상자는 비강(Nasal)을 통하여 검체를 채취하는 것을 특징으로 할 수 있다. In the present invention, in the case of the pulling test, a sample with a body temperature of 37.5° C. or lower is collected through the oral cavity, and a sample with a body temperature of 37.5° C. or higher and suspected symptoms is collected through the nasal cavity. can be done with
본 발명에 있어서, 상기 AQ-TOP™ COVID-19 Rapid Kit는 다음 (i)~(xi)에서 선택되는 SARS-CoV-2 유전자 증폭용 프라이머 쌍을 포함하는 것을 특징으로 할 수 있다:In the present invention, the AQ-TOP ™ COVID-19 Rapid Kit may be characterized by including a primer pair for SARS-CoV-2 gene amplification selected from the following (i) to (xi):
(i) 서열번호 1의 정방향 프라이머 및 서열번호 2의 역방향 프라이머로 구성되는 프라이머 쌍;(i) a primer pair consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2;
(ii) 서열번호 3의 정방향 프라이머 및 서열번호 4의 역방향 프라이머로 구성되는 프라이머 쌍;(ii) a primer pair consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4;
(iii) 서열번호 5의 정방향 프라이머 및 서열번호 6의 역방향 프라이머로 구성되는 프라이머 쌍;(iii) a primer pair consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6;
(iv) 서열번호 7의 정방향 프라이머 및 서열번호 8의 역방향 프라이머로 구성되는 프라이머 쌍;(iv) a primer pair consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8;
(v) 서열번호 9의 정방향 프라이머 및 서열번호 10의 역방향 프라이머로 구성되는 프라이머 쌍;(v) a primer pair consisting of a forward primer of SEQ ID NO: 9 and a reverse primer of SEQ ID NO: 10;
(vi) 서열번호 11의 정방향 프라이머 및 서열번호 12의 역방향 프라이머로 구성되는 프라이머 쌍;(vi) a primer pair consisting of a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12;
(vii) 서열번호 13의 정방향 프라이머 및 서열번호 14의 역방향 프라이머로 구성되는 프라이머 쌍;(vii) a primer pair consisting of a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 14;
(viii) 서열번호 15의 정방향 프라이머 및 서열번호 16의 역방향 프라이머로 구성되는 프라이머 쌍;(viii) a primer pair consisting of a forward primer of SEQ ID NO: 15 and a reverse primer of SEQ ID NO: 16;
(ix) 서열번호 17의 정방향 프라이머 및 서열번호 18의 역방향 프라이머로 구성되는 프라이머 쌍;(ix) a primer pair consisting of a forward primer of SEQ ID NO: 17 and a reverse primer of SEQ ID NO: 18;
(x) 서열번호 19의 정방향 프라이머 및 서열번호 20의 역방향 프라이머로 구성되는 프라이머 쌍; 및(x) a primer pair consisting of a forward primer of SEQ ID NO: 19 and a reverse primer of SEQ ID NO: 20; and
(xi) 서열번호 21의 정방향 프라이머 및 서열번호 22의 역방향 프라이머로 구성되는 프라이머 쌍.(xi) a primer pair consisting of a forward primer of SEQ ID NO: 21 and a reverse primer of SEQ ID NO: 22.
본 발명에 있어서, 상기 AQ-TOP™ COVID-19 Rapid Kit는 서열번호 23~30으로 구성된 군에서 선택되는 하나 이상의 서열로 표시되는 SARS-CoV-2 진단용 PNA 프로브를 포함하는 것을 특징으로 할 수 있다. In the present invention, the AQ-TOP ™ COVID-19 Rapid Kit may include a PNA probe for diagnosis of SARS-CoV-2 represented by one or more sequences selected from the group consisting of SEQ ID NOs: 23-30. .
본 발명의 검역시스템은 공항, 항만 또는 대규모 시설에서 수행하는 것을 특징으로 하는 검역시스템.The quarantine system of the present invention is a quarantine system, characterized in that it is performed at an airport, a port, or a large-scale facility.
본 발명의 일 양태에서, 상기 AQ-TOP™ COVID-19 Rapid Kit에 포함되는 프라이머 쌍은 SARS-CoV-2 바이러스의 ORF1ab 유전자 및 N 유전자를 증폭할 수 있는 프라이머 쌍이며, 상기 프라이머 쌍에 의해 증폭된 증폭산물은 서열번호 33 내지 서열번호 48의 PNA 프로브에 의하여 검출된다.In one aspect of the present invention, the primer pair included in the AQ-TOP™ COVID-19 Rapid Kit is a primer pair capable of amplifying the ORF1ab gene and N gene of SARS-CoV-2 virus, and amplified by the primer pair The amplified product is detected by the PNA probe of SEQ ID NOs: 33 to 48.
본 발명에 있어서 상기 PNA 프로브는 제한되지는 않으나 리포터 또는 소광자가 결합되는 것을 특징으로 할 수 있다.In the present invention, the PNA probe is not limited, but may be characterized in that a reporter or a quencher is bound.
본 발명의 프로브는 양 말단에 리포터와 리포터 형광을 소광할 수 있는 소광자의 형광 물질이 결합할 수 있으며, 인터컬레이팅(intercalating) 형광 물질을 포함할 수 있다. 상기 리포터는 FAM(6-carboxyfluorescein), HEX, Texas red, JOE, TAMRA, CY5, CY3, Alexa680 로 구성되는 군에서 선택되는 하나 이상일 수 있으며, 상기 소광자는 TAMRA(6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 또는 Dabcyl을 사용하는 것이 바람직하지만 이에 한정되는 것은 아니다. The probe of the present invention may bind to both ends of a reporter and a fluorescent material of a quencher capable of quenching reporter fluorescence, and may include an intercalating fluorescent material. The reporter may be one or more selected from the group consisting of FAM (6-carboxyfluorescein), HEX, Texas red, JOE, TAMRA, CY5, CY3, Alexa680, and the quencher is TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, It is preferred, but not limited to, BHQ2 or Dabcyl.
상기 인터컬레이팅 형광 물질은 아크리딘 호모다이머(Acridine homodimer) 및 이의 유도체, 아크리딘 오렌지(Acridine Orange) 및 이의 유도체, 7-아미노액티노마이신 D(7-aminoactinomycin D, 7-AAD) 및 이의 유도체, 액티노마이신 D(Actinomycin D) 및 이의 유도체, 에이씨엠에이(ACMA, 9-amino-6-chloro-2-methoxyacridine) 및 이의 유도체, 디에이피아이(DAPI) 및 이의 유도체, 디하이드로에티듐(Dihydroethidium) 및 이의 유도체, 에티듐 브로마이드(Ethidium bromide) 및 이의 유도체, 에티듐 호모다이머-1(EthD-1) 및 이의 유도체, 에티듐 호모다이머-2(EthD-2) 및 이의 유도체, 에티듐 모노아자이드(Ethidium monoazide) 및 이의 유도체, 헥시디움 아이오다이드(Hexidium iodide) 및 이의 유도체, 비스벤지마이드(bisbenzimide, Hoechst 33258) 및 이의 유도체, 호에크스트 33342(Hoechst 33342) 및 이의 유도체, 호에크스트 34580(Hoechst 34580) 및 이의 유도체, 하이드로옥시스티바미딘(hydroxystilbamidine) 및 이의 유도체, 엘디에스 751(LDS 751) 및 이의 유도체, 프로피디움 아이오다이드(Propidium Iodide, PI)와 이의 유도체 및 사이다이스(Cy-dyes) 유도체로 이루어진 군에서 선택될 수 있다.The intercalating fluorescent material is acridine homodimer and its derivatives, acridine orange and its derivatives, 7-aminoactinomycin D (7-AAD) and derivatives thereof, Actinomycin D and derivatives thereof, ACMA (9-amino-6-chloro-2-methoxyacridine) and derivatives thereof, DAPI and derivatives thereof, dihydroethidium (Dihydroethidium) and its derivatives, ethidium bromide and its derivatives, ethidium homodimer-1 (EthD-1) and its derivatives, ethidium homodimer-2 (EthD-2) and its derivatives, ethidium Ethidium monoazide and its derivatives, Hexidium iodide and its derivatives, bisbenzimide (Hoechst 33258) and its derivatives, Hoechst 33342 and its derivatives, ho Hoechst 34580 and its derivatives, hydroxystilbamidine and its derivatives, LDS 751 and its derivatives, propidium iodide (PI) and its derivatives and between It may be selected from the group consisting of dyes (Cy-dyes) derivatives.
본 발명에 있어서, 상기 AQ-TOP™ COVID-19 Rapid Kit는 대조물질; 이를 검출하기 위한 서열번호 31의 정방향 프라이머와 서열번호 32 역방향 프라이머의 프라이머 쌍, 서열번호 33의 정방향 프라이머와 서열번호 34의 역방향 프라이머의 프라이머 쌍; 및 서열번호 35~37의 PNA 프로브를 추가로 포함하는 것을 특징으로 할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the AQ-TOP™ COVID-19 Rapid Kit is a control material; a primer pair of a forward primer of SEQ ID NO: 31 and a reverse primer of SEQ ID NO: 32, a primer pair of a forward primer of SEQ ID NO: 33 and a reverse primer of SEQ ID NO: 34 for detecting this; And it may be characterized in that it further comprises a PNA probe of SEQ ID NOs: 35-37, but is not limited thereto.
본 발명에 있어서, 내부대조물질은 SARS-CoV-2의 존재 유무와 상관없이 임상 검체의 중합효소 연쇄반응의 성공 여부를 확인할 수 있는 유전자이면 제한없이 이용가능하나, 바람직하게는 homosapiens 종 Rnase P 유전자의 RNA 부위인 것을 특징으로 할 수 있다.In the present invention, the internal control material can be used without limitation as long as it is a gene that can confirm the success or failure of the polymerase chain reaction in a clinical specimen regardless of the presence or absence of SARS-CoV-2, but preferably a homosapiens species Rnase P gene It may be characterized as an RNA region of
본 발명에 있어서, 진단/분석 단계는 다음 단계로 수행하는 것이 바람직하다. In the present invention, the diagnosis/analysis step is preferably performed as the following step.
(a) 검체 시료로부터 RNA를 분리하는 단계;(a) isolating RNA from the specimen;
(b) 상기 프라이머 쌍을 이용하여 신종 코로나바이러스의 ORF1ab 유전자 및 N 유전자를 증폭시켜 증폭산물을 수득하는 단계;(b) amplifying the ORF1ab gene and the N gene of novel coronavirus using the primer pair to obtain an amplification product;
(c) 상기 증폭산물을 리포터 (reporter) 및 소광자 (quencher)가 결합되어 있는 상기 PNA 프로브와 혼성화시키는 단계 및 (c) hybridizing the amplification product with the PNA probe to which a reporter and a quencher are bound; and
(d) 형광검출 측정을 통해 신종 코로나바이러스(Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) 감염을 판정하는 단계.(d) determining the new coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) infection through fluorescence detection measurement.
본 발명에서, 상기 검체 시료는 DNA 또는 RNA 분자이며, 상기 분자는 이중가닥 또는 단일가닥 형체일 수 있다. 초기물질로써 핵산이 RNA 단일가닥인 경우, RNA 가닥을 complementary DNA(cDNA) 가닥으로 합성하는 역전사 반응단계를 거치는 것이 바람직하다. 이는 역전사효소(reverse trascriptase)를 사용하여 달성 될 수 있으며 이에 한정되는 것은 아니다. 또한 초기물질로서 핵산이 이중가닥인 경우, 두 가닥을 단일 가닥으로, 또는 부분적인 단일-가닥 형태로 만드는 것이 바람직하다. 가닥들을 분리하는 것으로 알려진 방법들은, 열, 알카리, 포름아미드, 우레아 및 글리콕살 처리, 효소적 방법(예, 헬리카아제 작용) 및 결합 단백질을 포함하나, 이에 한정되는 것은 아니다. 예를 들어, 가닥 분리는 80℃ 내지 105℃의 온도로 열처리하여 달성될 수 있다.In the present invention, the specimen sample is a DNA or RNA molecule, and the molecule may be in a double-stranded or single-stranded form. When the nucleic acid as an initial material is single-stranded RNA, it is preferable to undergo a reverse transcription reaction step of synthesizing the RNA strand into a complementary DNA (cDNA) strand. This may be achieved using, but not limited to, reverse trascriptase. In addition, when the nucleic acid as a starting material is double-stranded, it is preferable to make the two strands into a single-stranded or partially single-stranded form. Methods known to separate strands include, but are not limited to, heat, alkali, formamide, urea and glycoxal treatment, enzymatic methods (eg, helicase action) and binding proteins. For example, strand separation can be achieved by heat treatment at a temperature of 80°C to 105°C.
본 발명에서 상기 유전자는 제한되지는 않으나 SARS-CoV-2의 ORF1ab 유전자 또는 N 유전자일 수 있다.In the present invention, the gene is not limited, but may be the ORF1ab gene or N gene of SARS-CoV-2.
본 발명에 있어서, 분석 방법은 제한되지는 않으나, PCR, 역전사 중합효소 연쇄반응(Reverse transcription Real time PCR) 또는 등온증폭(Isothermal Amplification) PCR방법으로 수행하는 것을 특징으로 할 수 있다.In the present invention, the analysis method is not limited, but it may be characterized in that it is performed by PCR, reverse transcription real time PCR, or isothermal amplification PCR method.
본 발명에 있어서, 상기 b) 단계는 대조물질을 추가로 포함하는 것을 특징으로 할 수 있다.In the present invention, step b) may be characterized in that it further comprises a control material.
본 발명에 있어서, 상기 내부대조물질은 신종 코로나바이러스(SARS-CoV-2)의 존재 유무와 상관없이 임상 검체의 역전사 중합효소 연쇄반응의 성공 여부를 확인할 수 있는 유전자이면 제한없이 이용가능하나, 바람직하게는 homosapiens 종 RNase P 유전자의 RNA 부위인 것을 특징으로 할 수 있다.In the present invention, the internal control material can be used without limitation as long as it is a gene that can confirm the success of the reverse transcription polymerase chain reaction in a clinical sample regardless of the presence or absence of the novel coronavirus (SARS-CoV-2), but preferably It can be characterized as an RNA region of the homosapiens species RNase P gene.
본 발명의 '표적 핵산', '합성 DNA' 또는 '인공합성 올리고'는 검출 여부를 판별하고자 하는 핵산 서열을 의미하며, 생리ㆍ생화학적 기능을 가지는 단백질을 코딩하는 '표적 유전자'의 핵산 서열의 특정 부위를 포함하고, 혼성화, 어닐링 또는 증폭 조건 하에서 프라이머 또는 프로브와 어닐링 또는 혼성화된다.The 'target nucleic acid', 'synthetic DNA' or 'artificial oligo' of the present invention means a nucleic acid sequence to be detected, and the nucleic acid sequence of a 'target gene' encoding a protein having physiological and biochemical functions. It contains a specific site and is annealed or hybridized with a primer or probe under hybridization, annealing or amplification conditions.
본 발명의 '혼성화'는 상보적인 단일가닥 핵산들이 이중-가닥 핵산을 형성하는 것을 의미한다. 혼성화는 2개의 핵산 가닥 간의 상보성이 완전할 경우(perfect match) 일어나거나 또는 일부 부정합(mismatch) 염기가 존재하여 도 일어날 수 있다. 혼성화에 필요한 상보성의 정도는 혼성화 조건에 따라 달라질 수 있으며, 특히 온도에 의하여 조절될 수 있다.'Hybridization' in the present invention means that complementary single-stranded nucleic acids form double-stranded nucleic acids. Hybridization may occur when complementarity between two nucleic acid strands is perfect (perfect match) or even when some mismatched bases are present. The degree of complementarity required for hybridization may vary depending on hybridization conditions, and in particular may be controlled by temperature.
본 발명의 리포터 및 소광자가 포함된 PNA 프로브는 표적 핵산과 혼성화된 후 형광 신호가 발생하며, 이를 분석을 통하여 표적 핵산의 유무를 검출할 수 있다.The PNA probe containing the reporter and quencher of the present invention generates a fluorescent signal after hybridization with the target nucleic acid, and the presence or absence of the target nucleic acid can be detected through analysis.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1: SARS-CoV-2 유전자 증폭을 위한 프라이머 및 검출용 프로브 제작Example 1: Preparation of primers and detection probes for SARS-CoV-2 gene amplification
신종 코로나바이러스의 표적물질(ORF1ab 유전자 및 N 유전자)에 대한 시간 역전사중합효소 반응을 위해, 하기 표 3와 같이 서열번호 1 ~ 18의 프라이머를 제작하였다. For the time reverse transcriptase reaction of the novel coronavirus target material (ORF1ab gene and N gene), primers of SEQ ID NOs: 1 to 18 were prepared as shown in Table 3 below.
Figure PCTKR2021008506-appb-img-000003
Figure PCTKR2021008506-appb-img-000003
Figure PCTKR2021008506-appb-img-000004
Figure PCTKR2021008506-appb-img-000004
신종 코로나바이러스의 특이적 증폭분석을 위하여 서열번호 23 내지 서열번호 30의 PNA 프로브를 제작하였으며, 다중 증폭을 위해 프로브 서열 양 끝에 각각 형광과 소광자를 결합시켰다(표 4).For the specific amplification analysis of novel coronavirus, PNA probes of SEQ ID NOs: 23 to 30 were prepared, and fluorescence and quenchers were respectively coupled to both ends of the probe sequence for multiple amplification (Table 4).
Figure PCTKR2021008506-appb-img-000005
Figure PCTKR2021008506-appb-img-000005
실시예 2: 대조물질 증폭을 위한 프라이머 제작Example 2: Preparation of primers for control amplification
진단 키트 내 대조물질(IC, Internal Control) 도입을 위하여 Homosapiens의 Rnase P 유전자의 RNA 150bp 부위를 표적핵산으로 사용하여 이를 검출 할 수 있는 프라이머를 제작하였으며(표 5), 상기 대조물질의 증폭산물을 검출할 수 있는 PNA 프로브를 제작하였다(표 6).In order to introduce a control material (IC, Internal Control) into the diagnostic kit, a primer capable of detecting this was prepared using the RNA 150bp region of the Rnase P gene of Homosapiens as a target nucleic acid (Table 5), and the amplification product of the control material was prepared. A detectable PNA probe was prepared (Table 6).
Figure PCTKR2021008506-appb-img-000006
Figure PCTKR2021008506-appb-img-000006
Figure PCTKR2021008506-appb-img-000007
Figure PCTKR2021008506-appb-img-000007
본 발명에 따르면, AQ-TOPTM COVID-19 Plus 키트를 이용한 4S(Seasun Smart Shield System)를 적용할 경우, 코로나 19 감염여부를 신속하고, 정확하게 판별이 가능하여 인구의 이동이 잦은 공항, 항만 등에서의 신속검역, 대규모 집회 및 집단 감염 지역에서의 전수검사에 효과적이다.According to the present invention, when the 4S (Seasun Smart Shield System) using the AQ-TOP TM COVID-19 Plus kit is applied, it is possible to quickly and accurately determine whether the person is infected with COVID-19, so that the population moves frequently in airports, ports, etc. It is effective for rapid quarantine, large-scale gatherings, and total inspection in areas with mass infection.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시태양일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.
전자파일 첨부하였음.An electronic file is attached.

Claims (17)

  1. SARS-CoV-2 검출용 AQ-TOP™ COVID-19 Rapid Kit를 이용한 코로나 19 검역시스템.Corona 19 quarantine system using AQ-TOP™ COVID-19 Rapid Kit for SARS-CoV-2 detection.
  2. 제1항에 있어서, 풀링 테스트(pooling test), 단일 테스트(single test) 또는 긴급 테스트(emergency test) 인 것을 특징으로 하는 검역시스템.The quarantine system according to claim 1, characterized in that it is a pooling test, a single test, or an emergency test.
  3. 제1항에 있어서, 상기 검역시스템은 공항, 항만, 대규모 시설 또는 집단감염 지역에서 수행하는 것을 특징으로 하는 검역시스템.The quarantine system according to claim 1, wherein the quarantine system is performed at an airport, a port, a large-scale facility, or a cluster infection area.
  4. 제2항에 있어서, 상기 풀링 테스트(pooling test)는 신원확인 단계; 검체 채취 단계; 바이러스 분리 단계; 및 진단/분석 단계를 포함하는 것을 특징으로 하는 검역시스템.3. The method of claim 2, wherein the pooling test comprises: an identification step; sampling step; virus isolation step; and a diagnosis/analysis step.
  5. 제4항에 있어서, 상기 검체 채취 단계는 3~4명의 검체를 하나의 튜브에 넣어 검사하는 풀링(pooling) 테스트로 진행하는 것을 특징으로 하는 검역시스템.5. The quarantine system according to claim 4, wherein the sample collection step proceeds with a pooling test in which 3 to 4 samples are put into one tube and tested.
  6. 제4항에 있어서, 상기 검체 채취는 구강(Oral) 또는 비강(Nasal)을 통하여 채취하는 것을 특징으로 하는 검역시스템.The quarantine system according to claim 4, wherein the sample is collected through the oral cavity or the nasal cavity.
  7. 제4항에 있어서, 상기 검체 채취 단계는 체온이 37.5℃ 이하의 유증상자는 구강(Oral)을 통하여 검체를 채취하고, 37.5℃ 이상 및 의심증상자는 비강(Nasal)을 통하여 검체를 채취하는 것을 특징으로 하는 검역시스템.[Claim 5] The method of claim 4, wherein in the sample collection step, a sample with a body temperature of 37.5° C. or lower is collected through the oral cavity, and a sample with a body temperature of 37.5° C. or higher and suspected symptoms is collected through the nasal cavity. quarantine system.
  8. 제4항에 있어서, 상기 진단/분석 단계는 AQ-TOP™ COVID-19 Rapid Kit를 이용하여 PCR로 수행하는 것을 특징으로 하는 검역시스템.The quarantine system according to claim 4, wherein the diagnosis/analysis step is performed by PCR using AQ-TOP™ COVID-19 Rapid Kit.
  9. 제2항에 있어서, 상기 단일 테스트(single test)는 신원확인 단계; 검체 채취 단계; 바이러스 분리 단계; 및 진단/분석 단계를 포함하는 것을 특징으로 하는 검역시스템.3. The method of claim 2, wherein the single test comprises: an identification step; sampling step; virus isolation step; and a diagnosis/analysis step.
  10. 제9항에 있어서, 상기 검체 채취 단계는 1명의 검체를 하나의 튜브에 넣어 검사하는 단일 테스트로 진행하는 것을 특징으로 하는 검역시스템.The quarantine system according to claim 9, wherein the sample collection step is performed as a single test in which one sample is put into one tube and tested.
  11. 제9항에 있어서, 상기 진단/분석 단계는 AQ-TOP™ COVID-19 Rapid Kit를 이용하여 등온증폭 (Isothermal Amplification) PCR로 수행하는 것을 특징으로 하는 검역시스템.The quarantine system according to claim 9, wherein the diagnosis/analysis step is performed by isothermal amplification PCR using AQ-TOP™ COVID-19 Rapid Kit.
  12. 제2항에 있어서, 상기 긴급 테스트(emergency test)는 신원확인 단계; 검체 채취 단계; 바이러스 분리 단계; 및 진단/분석 단계를 포함하는 것을 특징으로 하는 검역시스템.According to claim 2, wherein the emergency test (emergency test) the identification step; sampling step; virus isolation step; and a diagnosis/analysis step.
  13. 제12항에 있어서, 상기 긴급 테스트(emergency test)는 풀링테스트에서 양성판별자에 대하여 시행하는 것을 특징으로 하는 검역시스템.The quarantine system according to claim 12, wherein the emergency test is performed on positive persons in a pooling test.
  14. 제12항에 있어서, 상기 검체 채취 단계는 1명의 검체를 하나의 튜브에 넣어 검사하는 단일 테스트로 진행하는 것을 특징으로 하는 검역시스템.The quarantine system according to claim 12, wherein the sample collection step is performed as a single test in which one sample is put into one tube and tested.
  15. 제12항에 있어서, 상기 진단/분석 단계는 AQ-TOP™ COVID-19 Rapid Kit를 이용하여 등온증폭 (Isothermal Amplification) PCR로 수행하는 것을 특징으로 하는 검역시스템.The quarantine system according to claim 12, wherein the diagnosis/analysis step is performed by isothermal amplification PCR using the AQ-TOP™ COVID-19 Rapid Kit.
  16. 제1항에 있어서, 상기 AQ-TOP™ COVID-19 Rapid Kit는 다음 (i)~(xi)에서 선택되는 SARS-CoV-2 유전자 증폭용 프라이머 쌍을 포함하는 것을 특징으로 하는 검역시스템:The quarantine system according to claim 1, wherein the AQ-TOP™ COVID-19 Rapid Kit includes a pair of primers for amplifying the SARS-CoV-2 gene selected from the following (i) to (xi):
    (i) 서열번호 1의 정방향 프라이머 및 서열번호 2의 역방향 프라이머로 구성되는 프라이머 쌍;(i) a primer pair consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2;
    (ii) 서열번호 3의 정방향 프라이머 및 서열번호 4의 역방향 프라이머로 구성되는 프라이머 쌍;(ii) a primer pair consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4;
    (iii) 서열번호 5의 정방향 프라이머 및 서열번호 6의 역방향 프라이머로 구성되는 프라이머 쌍;(iii) a primer pair consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6;
    (iv) 서열번호 7의 정방향 프라이머 및 서열번호 8의 역방향 프라이머로 구성되는 프라이머 쌍;(iv) a primer pair consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8;
    (v) 서열번호 9의 정방향 프라이머 및 서열번호 10의 역방향 프라이머로 구성되는 프라이머 쌍;(v) a primer pair consisting of a forward primer of SEQ ID NO: 9 and a reverse primer of SEQ ID NO: 10;
    (vi) 서열번호 11의 정방향 프라이머 및 서열번호 12의 역방향 프라이머로 구성되는 프라이머 쌍;(vi) a primer pair consisting of a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12;
    (vii) 서열번호 13의 정방향 프라이머 및 서열번호 14의 역방향 프라이머로 구성되는 프라이머 쌍;(vii) a primer pair consisting of a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 14;
    (viii) 서열번호 15의 정방향 프라이머 및 서열번호 16의 역방향 프라이머로 구성되는 프라이머 쌍;(viii) a primer pair consisting of a forward primer of SEQ ID NO: 15 and a reverse primer of SEQ ID NO: 16;
    (ix) 서열번호 17의 정방향 프라이머 및 서열번호 18의 역방향 프라이머로 구성되는 프라이머 쌍;(ix) a primer pair consisting of a forward primer of SEQ ID NO: 17 and a reverse primer of SEQ ID NO: 18;
    (x) 서열번호 19의 정방향 프라이머 및 서열번호 20의 역방향 프라이머로 구성되는 프라이머 쌍; 및(x) a primer pair consisting of a forward primer of SEQ ID NO: 19 and a reverse primer of SEQ ID NO: 20; and
    (xi) 서열번호 21의 정방향 프라이머 및 서열번호 22의 역방향 프라이머로 구성되는 프라이머 쌍.(xi) a primer pair consisting of a forward primer of SEQ ID NO: 21 and a reverse primer of SEQ ID NO: 22.
  17. 제1항에 있어서, 상기 AQ-TOP™ COVID-19 Rapid Kit는 서열번호 23~30으로 구성된 군에서 선택되는 하나 이상의 서열로 표시되는 SARS-CoV-2 진단용 PNA 프로브를 포함하는 것을 특징으로 하는 검역시스템. The quarantine according to claim 1, wherein the AQ-TOP™ COVID-19 Rapid Kit comprises a PNA probe for diagnosis of SARS-CoV-2 represented by one or more sequences selected from the group consisting of SEQ ID NOs: 23-30. system.
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