KR102109196B1 - LAMP composition for detecting 2019 novel Coronavirus and uses thereof - Google Patents

Abstract

The present invention relates to a loop-mediated isothermal amplification (LAMP) composition for detecting the 2019 novel coronavirus and a use thereof. A method for detecting the 2019 novel coronavirus using the LAMP composition of the present invention can confirm a molecular diagnosis test result within an hour in the field only with a real-time isothermal amplification instrument without separate nucleic acid extraction and purification processes from a sample, thereby usefully used for a field diagnosis test, etc.

Description

LAMP composition for 2019 new Coronavirus detection and its use {LAMP composition for detecting 2019 novel Coronavirus and uses thereof}

The present invention relates to 2019 LOP (Loop-mediated isothermal amplification) composition for detection of new coronavirus and its use.

The new coronavirus (2019 novel Coronavirus, 2019-nCoV) is the cause of the new coronavirus infection (Wuhan pneumonia) that occurred in December 2019, and is one of seven coronaviruses that infect humans. The new coronavirus has been reported to spread when the droplets (droplets) of the infected person penetrate the respiratory tract or mucous membranes of the eyes, nose, and mouth, and when infected, the fever (37.5 degrees) after an incubation period of about 2 to 14 days (estimated) And respiratory symptoms such as coughing or dyspnea, and pneumonia are the main symptoms, but asymptomatic infection cases are also rare. The World Health Organization (WHO) said on January 24, 2020, the spread of the new coronavirus was lower than that of SARS (severe acute respiratory syndrome), but higher than that of MERS (MERS, Middle East Respiratory Syndrome).

According to the report from the Korea Centers for Disease Control and Prevention, the gene sequence of the new coronavirus showed the highest homology (89.1%) to that of the bat-derived similar coronavirus, with 50% with MERS and 77.5% with SARS. Coronavirus is classified into four genus, such as Alpha, Beta, Gamma, and Delta, and the new coronavirus is classified as belonging to the Beta group. There are seven human infectious coronaviruses identified so far, including HCoV 229E, HCoV NL63, HCoV OC43, HCoV HKU1, SARS-CoV, MERS-CoV, and 2019-nCoV.

Currently, the confirmation of the new corona is achieved through the conformation of the sequencing with the conventional coronavirus test (Conventional PCR). This is a coronal virus type (pancorona test) for suspicious patients, and if a positive response occurs, the virus gene sequence from the patient's sample is analyzed and tested. It takes about 1 to 2 days. However, from January 31, 2020, a new test method targeting only the new coronavirus, the so-called 'Real Time PCR', was applied by the Centers for Disease Control (including the National Incheon Airport Quarantine Station) and 18 health and environmental research institutes nationwide. The core of this test method is a 'reagent kit' that can identify and diagnose a new coronavirus rather than the entire coronavirus family, such as a plate corona test, and the results can be confirmed within 6 hours of the test.

On the other hand, Korean Registered Patent No. 2018079 discloses a 'primer set for detecting MERS coronavirus and its use', and Korean Registered Patent No. 1100436 discloses a 'Primary for detecting SARS coronavirus, a method for detecting SARS coronavirus using the same, and Kit 'is disclosed, but the' 2019 new coronavirus detection LAMP composition and its use 'has not been described.

The present invention was derived by the above-mentioned needs, and the present inventors prepared a primer set for isothermal amplification specific to the N gene encoding the nucleocapsid protein of the 2019 new coronavirus, and then infected with the new coronavirus (right pneumonia). As a result of performing reverse transcriptal loop-mediated isothermal amplification (RT-LAMP) on a sample of, a nucleic acid of a new coronavirus (2019 novel Coronavirus) can be accurately detected in the sample without a non-specific reaction. By confirming the existence, the present invention was completed.

In order to solve the above problems, the present invention is for reverse transcriptional loop-mediated isothermal amplification for detecting 2019 new Coronavirus containing oligonucleotide primers of SEQ ID NOs: 1 to 6. Provide a primer set.

In addition, the present invention provides a composition for detecting a new coronavirus 2019 comprising the primer set for reverse transcription mediated isothermal amplification.

In addition, the present invention provides a kit for detecting a new coronavirus 2019 comprising the primer set for reverse transcription ring-mediated isothermal amplification and a reagent for performing ring-mediated isothermal amplification.

In addition, the present invention comprises the steps of separating total RNA from biological samples isolated from patients suspected of 2019 new coronavirus infection; Amplifying the target sequence by using the isolated total RNA as a template, and using the primer set for reverse transcription loop isothermal amplification of the present invention, by reverse transcriptional loop-mediated isothermal amplification; And detecting the product of the amplification step; provides a method for detecting a 2019 new coronavirus (2019 novel Coronavirus).

In addition, the present invention comprises the steps of pre-treating a biological sample isolated from a patient suspected of 2019 new coronavirus infection with a lysis buffer; Amplifying the target sequence by a reverse transcription loop isothermal amplification reaction using the reverse transcription mediated isothermal amplification primer set of the sample pre-treated with the lysis buffer; And detecting the product of the amplification step; provides a method for detecting a 2019 new coronavirus (2019 novel Coronavirus).

The LAMP composition of the present invention and the 2019 new coronavirus detection method using the same can be confirmed with the nasal swab solution and the simple real-time isothermal amplification device without genetic extraction. Although the PCR test method takes about 6 hours, it can provide test results quickly, so it can be useful for the diagnosis of 2019 new coronavirus infection.

Figure 1 shows the location of the base sequence region and the LAMP primer used in the production of LAMP primers specific to the nucleocapsid phosphoprotein encoding gene of the right-handed pneumonia virus used in the present invention.
FIG. 2 shows the results of RT-LAMP performed using the LAMP composition of the present invention as a BioRad CFX 96 Real time PCR instrument, and specifically amplifies only the 2019-nCoV clinical sample at 22 cycles.
FIG. 3 shows the results of RT-LAMP performed using the LAMP composition of the present invention as a portable real-time isothermal amplifier, and specifically amplifies only 2019-nCoV clinical samples from 16 minutes onwards.
4 is a result of Direct RT-LAMP performed using the LAMP composition of the present invention as a portable real-time isothermal amplifier, and developed by the present inventor without a separate nucleic acid extraction step of a clinical sample (sample) obtained by a nose swab method This is the result of isothermal amplification using a lysis buffer. From 42 minutes onwards, only 2019-nCoV clinical specimens are specifically amplified.
FIG. 5 shows the results of homology analysis between coronaviruses of the gene encoding the nucleocapsid protein ( N gene).

In order to achieve the object of the present invention, the present invention is reverse transcriptional loop-mediated isothermal amplification for detecting 2019 new Coronavirus (2019 novel Coronavirus) comprising the oligonucleotide primers of SEQ ID NOs: 1 to 6. , RT-LAMP).

The primer set for RT-LAMP for detecting the 2019 new coronavirus according to the present invention is GenBank accession no. MN908947.3, Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, includes a set of oligonucleotide primers of SEQ ID NOs: 1-6, specific for the gene encoding the nucleocapsid protein (SEQ ID NO: 7) in the complete genome.

The primer set according to the present invention extracts and purifies nucleic acids from a sample (sample), and performs reverse transcription and LAMP reactions simultaneously by mixing reverse transcriptase and polymerase in a single tube with RNA as a template. It can also be used in the reverse transcription ring-mediated isothermal amplification (RT-LAMP) method, or can be used in a method of performing a secondary LAMP method using cDNA made by first performing reverse transcription on RNA. 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 lysis buffer and the sample characterized in that it consists of 30 to 500 mM Tris of pH 6.0 to 7.0. It can be used in a direct RT-LAMP reaction method that directly performs LAMP without separate nucleic acid extraction and purification, but is not limited thereto.

In the present specification, the term 'Loop-mediated isothermal amplification (LAMP)' means a method capable of performing an amplification reaction under isothermal conditions, unlike the conventional polymerase chain reaction (PCR) method. Basically, 4 primers (F3, B3, FIP, BIP) are required for the LAMP reaction, and 2 primers (LF, LB) are added to improve the reaction rate, resulting in the final 6 different base sequences. The resulting oligonucleotide primer is necessary for the reaction.

The four basic primers are composed of two outer primers and two inner primers, and the outer primers are forward outer (F3) primers and reverse outer (B3) primers. It is composed of and serves to release the DNA double strand during the non-cyclic step of the reaction. The inner primer is composed of two types of forward inner primer (FIP) and backward inner primer (BIP), and the forward and reverse sequencing sequences make loops essential for isothermal amplification reactions. It consists of the corresponding nucleotides. The additional two primers are composed of two forward loop (LoopF) primers and two reverse loop (LoopB) primers, and are attached to a base sequence to which an inner primer does not bind to effect ring-mediated isothermal amplification. Accelerate.

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 F3, which is a forward outer primer, and the primer of SEQ ID NO: 2 is B3, which is a reverse outer primer, and a primer of SEQ ID NO: 3 Is a forward internal primer FIP, the primer of SEQ ID NO: 4 is a reverse internal 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.

The primer of the present invention may include oligonucleotides consisting of segments 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 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, or 37 or more consecutive 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.

As used herein, the term "primer" refers to a single-stranded oligonucleotide sequence complementary to the nucleic acid strand to be cloned, and may serve as a starting point for the synthesis of primer extension products. The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the desired DNA or RNA target complexity, as well as the conditions of primer use, such as temperature and ionic strength.

In the present invention, the oligonucleotide used as a primer may also include a nucleotide analog, for example, phosphorothioate, alkylphosphorothioate or peptide nucleic acid, or And an intercalating agent.

In addition, in order to increase the convenience of detection, each primer of the present invention may be modeled as a label for detection. The label for detection may be a compound, a biomolecule or a biomolecule analog, or the like capable of confirming the density, concentration, amount, etc. of amplification products in a conventional manner by connecting, binding, or attaching to a primer, but is not limited thereto, 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 2019 new coronavirus (2019 novel Coronavirus) including a primer set for reverse transcription ring-mediated isothermal amplification for detecting the 2019 new coronavirus. 2019 new species coronavirus detection composition according to the present invention GenBank accession no. MN908947.3, Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, includes a set of oligonucleotide primers of SEQ ID NOs: 1-6, specific for the gene encoding the nucleocapsid protein (SEQ ID NO: 7) in the complete genome.

The present invention also provides a kit for detecting 2019 new coronavirus (2019 novel Coronavirus) comprising a primer set for reverse transcription ring-mediated isothermal amplification and a reagent for performing ring-mediated isothermal amplification reaction for detecting the 2019 new coronavirus do.

2019 new coronavirus detection kit according to the present invention is GenBank accession no. MN908947.3, Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, includes a set of oligonucleotide primers of SEQ ID NOs: 1-6, specific for the gene encoding the nucleocapsid protein (SEQ ID NO: 7) in the complete genome.

In addition, the 2019 new coronavirus detection kit according to the present invention may further include, but is not limited to, a fluorescently labeled probe capable of binding to the product amplified by the ring-mediated isothermal amplification primer set.

In the kit of the present invention, the reagent for performing the ring-mediated isothermal amplification reaction may include DNA polymerase, dNTPs and buffers. Since the new coronavirus 2019 is an RNA virus, a reverse transcription ring-mediated isothermal amplification method using a reverse transcription process must be used for amplification. Therefore, the kit of the present invention may further include a reverse transcriptase.

The 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 an existing ring-mediated isothermal amplification method (LAMP) for reverse transcription ring-mediated isothermal amplification (RT-LAMP). GspSSD DNA polymerase, which has a faster amplification ability and a strong reverse transcriptase ability, may be used instead of the Bst polymerase, but is not limited thereto.

The new coronavirus detection kit of 2019 of the present invention may preferably be a kit for reverse transcription ring-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, so that the diagnosis is faster compared to the conventional LAMP diagnostic method that requires the step of extracting and purifying nucleic acids from the sample. There are possible features.

When the kit of the present invention is for Direct RT-LAMP, the kit further includes a lysis buffer, and the lysis buffer preferably contains 0.1 to 1.5% (v / v) of Triton X-100, pH 6.0. 30 to 500 mM Tris of ∼7.0, more preferably 300 to 500 mM Tris of pH 6.5, including 0.8 to 1.2% (v / v) of Triton X-100, even more preferably May be 400 mM Tris of pH 6.5, including 1% (v / v) of Triton X-100, but is not limited thereto.

In addition, the kit of the present invention may further include a user guide describing optimal conditions for performing the reaction. The handbook is a printout that describes how to use the kit, for example, how to prepare a buffer, and the reaction conditions presented. The guide includes brochures in the form of brochures or flyers, labels attached to the kit, and descriptions on the surface of the package containing the kit. In addition, the handbook includes information that is disclosed or provided through electronic media, such as the Internet.

The present invention also provides a method for detecting a new 2019 coronavirus using a primer set for reverse transcription mediated isothermal amplification for detecting the 2019 new coronavirus containing 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.

2019 new species coronavirus detection method according to an embodiment of the present invention,

Separating total RNA from biological samples isolated from patients suspected of 2019 new coronavirus infection;

Amplifying the target sequence by using the isolated total RNA as a template, and using the primer set for reverse transcription loop isothermal amplification of the present invention, by reverse transcriptional loop-mediated isothermal amplification; And

Detecting a product of the amplification step; may include, but is not limited to.

The method of the present invention includes the step of isolating total RNA from biological samples isolated from suspected patients with 2019 new coronavirus infection. The sample may be, for example, a human sample collected (nasal swab), but is not limited thereto, and is not limited if it is a biological sample separated from suspected patients with 2019 new coronavirus infection. The method for separating total RNA from the sample may use any method known in the art, for example, a phenol extraction method.

Another detection method of the 2019 new coronavirus according to an embodiment of the present invention,

Pre-processing biological samples separated from suspected patients with 2019 new coronavirus infection with lysis buffer;

Amplifying the target sequence by a reverse transcription loop isothermal amplification reaction using the reverse transcription mediated isothermal amplification primer set of the sample pre-treated with the lysis buffer; And

Detecting a product of the amplification step; may include, but is not limited to.

Pre-treatment of the sample according to the detection method of the present invention (e.g., a nose swab sample or isolated human blood) with a lysis buffer is preferably performed within 5 minutes of mixing the sample and the lysis buffer in a volume ratio of 1: 9. It 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) Triton X-100, more preferably 0.8-1.2% (v / It may be 300 ~ 500 mM Tris of pH 6.5, including Triton X-100 of v), more preferably 400 mM Tris of pH 6.5, containing 1% (v / v) of Triton X-100. It may be, but is not limited thereto.

In the method for detecting new coronavirus in 2019 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 20 ° C for 50 minutes to 65 minutes. However, it is not limited thereto.

The method for detecting a new coronavirus in 2019 of the present invention includes the step of detecting the product of the reverse transcription loop mediation isothermal amplification step. Detection of the amplification product is fluorescence measurement using SYBR Green I, color change of indicators such as phenol red, turbidity, sediment generation, 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, DNA laddering, but is not limited thereto.

In addition, using a device that can detect fluorescent dyes in the reaction solution in real time, check isothermal amplification in real time without adding electrophoresis or adding SYBR Green I after reaction, and amplifying the target sequence through annealing peak. You can also check whether it is.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example 1. 2019 LAMP primer set design for detection of new coronavirus

To produce a primer set for ring-mediated isothermal amplification (LAMP) for the detection of 2019 novel Coronavirus, GenBank accession no. Information of the gene encoding the nucleocapsid protein (28,274 to 29,533, SEQ ID NO: 7) was obtained from the Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, a complete genome, MN908947.3. Primer to be used for LAMP was designed using OptiGene's LAMP Design software, and the final designed LAMP primer for 2019 detection of new coronavirus is the 401 ~ 700th nucleotide sequence from the nucleocapsid protein coding gene sequence consisting of the nucleotide sequence of SEQ ID NO: 7 Target sites, and the location 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 from the 401 to 700 nucleotide sequence region of various 2019 coronavirus base sequences (GenBank accession no: LC521925, LR757995, LR757996, LR757997, LR757998, MN938384, MN975262, Aligned 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.

In addition, previous studies have shown that other human infectious coronaviruses (HCoV 229E, HCoV OC43, HCoV HKU1, HCoV NL63 and MERS-) when targeting the gene encoding the nucleocapsid protein of 2019 new coronavirus ( N gene) CoV) was confirmed as undetectable results (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 Since the base sequence and information targeted by the F3 (SEQ ID NO: 1), B3 (SEQ ID NO: 2), and BIP primer (SEQ ID NO: 3) of the invention are different (FIG. 5), the dumbbell structure of the LAMP is not formed, resulting in a normal LAMP reaction. It was not expected.

Example 2. 2019 new coronavirus Detection capability analysis

In order to analyze the detection ability of 2019 new coronavirus using the produced LAMP primer, RT-LAMP using a clinical sample was performed. 2019 new coronavirus infection clinical samples were provided and used by the National Capital University Hospital, and induced similar clinical symptoms (cough and / or fever) to confirm whether the LMAP primer set of the present invention specifically binds to the 2019 new coronavirus Adenovirus type 55, Haemophilus Influenzae , Streptococcus pneumoniae , Mycoplasma pneumonia , Severe fever with thrombocytopenia syndrome virus, SFTS virus), malaria, and Tstsugamushi disease (Scrub typhus) infection clinical specimens (respiratory specimens) were provided by the Army Training Center and used as comparative groups.

RT-LAMP (Reverse transcriptional Loop-mediated Isothermal Amplification) reaction is performed with 2 μl of template RNA, 1 μl of Bst polymerase (8 U), 2.5 μl of 10 × buffer, 1.5 μl of 25 mM dNTP, 1 μl of 10 mM MgSO ₄ , 5 μl of 5M betaine. , Reverse Transcriptase 0.1µl (10U), 6 primers (5pmol each for F3 and B3, 40pmol each for FIP and BIP, 10pmol each for LoopF and LoopB) 1µl each, 25 × SYBR Green I 1µl and distilled water ( up to ~ 25µl). In order to check the results of the isothermal amplification products in real time, an isothermal amplification reaction was performed at 65 ° C. for 20 minutes using a BioRad CFX 96 Real time PCR instrument or a portable Isothermal Fluorescence PCR instrument Gene-8C (ALLSHENG). Fluorescence signals were measured every 30 seconds.

As a result of RT-LAMP performed on a real time PCR device, amplification began to occur only in the 2019 new coronavirus infection sample from 22 cycles, and it was confirmed that the 2019 new coronavirus infection sample was specifically detected in 30 cycles (FIG. 2). . In addition, as a result of RT-LAMP performed on a portable isothermal amplification device, an amplification product was confirmed only in the sample of new coronavirus infection from 16 minutes after the reaction time elapsed, and until 20 minutes after the reaction was terminated, H. influenzae ( H. influenzae ), Streptococcus It was found that no amplification products were found in pneumoniae ( S. pneumoniae ) and mycoplasma pneumoniae ( M. pneumonia ) 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 2019 new coronavirus.

Example 3. Direct RT-LAMP analysis ability evaluation

After mixing 50 μl of the sample obtained by nasal swab with nasal swab and 450 μl of the lysis buffer (400 mM Tris, pH 6.5, 1% Triton X-100) developed by the present inventors, mix through vortexing, and then Take 2 μl of the mixture, LAMP premix (1 μl of Bst polymerase (8 U), 2.5 μl of 10 × buffer, 1.5 μl of 25 mM dNTPs, 1 μl of 10 mM MgSO ₄ , 5 μl of 5M betaine), 6 primers (F3 and B3 each) 5 pmol, 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) are added, Isothermal Fluorescence PCR, Gene-8C (ALLSHENG, China) to perform an isothermal amplification reaction at 65 ° C for 50 minutes. Fluorescence signals were measured every 15 seconds.

As a result, although the detection time in which the amplification product was confirmed was delayed as compared with 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 reaction time from 42 minutes to 2019 new coronavirus infection The amplification products are confirmed only in the sample, and in the samples infected with H. influenza ( H. Influenzae ) and Mycoplasma pneumoniae ( M. pneumonia ), the amplification products are not confirmed until the end of the reaction (FIG. 4), Direct RT according to the present invention -It was found that 2019 new coronavirus can be accurately detected through LAMP. Through the above results, it is expected that the primer set of the present invention and the method for detecting the new coronavirus 2019 using the same will be very useful for diagnosing the infection of the 2019 new coronavirus (Wuhan pneumonia).

<110> The Armed Forces Medical Command <120> LAMP composition for detecting 2019 novel Coronavirus and uses          thereof <130> PN20031 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tcctgctaac aatgctgc 18 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tcaatctgtc aagcagcag 19 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aacgagaaga ggcttgactg ctcaaggaac aacattgcca 40 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ctcatcacgt agtcgcaaca gtattgccag ccattctagc 40 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ccttctgcgt agaagcctt 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aattcaactc caggcagca 19 <210> 7 <211> 1260 <212> DNA <213> Unknown <220> <223> Wuhan seafood market pneumonia virus <400> 7 atgtctgata atggacccca aaatcagcga aatgcacccc gcattacgtt tggtggaccc 60 tcagattcaa ctggcagtaa ccagaatgga gaacgcagtg gggcgcgatc aaaacaacgt 120 cggccccaag gtttacccaa taatactgcg tcttggttca ccgctctcac tcaacatggc 180 aaggaagacc ttaaattccc tcgaggacaa ggcgttccaa ttaacaccaa tagcagtcca 240 gatgaccaaa ttggctacta ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa 300 atgaaagatc tcagtccaag atggtatttc tactacctag gaactgggcc agaagctgga 360 cttccctatg gtgctaacaa agacggcatc atatgggttg caactgaggg agccttgaat 420 acaccaaaag atcacattgg cacccgcaat cctgctaaca atgctgcaat cgtgctacaa 480 cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt 540 caagcctctt ctcgttcctc atcacgtagt cgcaacagtt caagaaattc aactccaggc 600 agcagtaggg gaacttctcc tgctagaatg gctggcaatg gcggtgatgc tgctcttgct 660 ttgctgctgc ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa 720 caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa 780 aaacgtactg ccactaaagc atacaatgta acacaagctt tcggcagacg tggtccagaa 840 caaacccaag gaaattttgg ggaccaggaa ctaatcagac aaggaactga ttacaaacat 900 tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960 ggcatggaag tcacaccttc gggaacgtgg ttgacctaca caggtgccat caaattggat 1020 gacaaagatc caaatttcaa agatcaagtc attttgctga ataagcatat tgacgcatac 1080 aaaacattcc caccaacaga gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa 1140 gccttaccgc agagacagaa gaaacagcaa actgtgactc ttcttcctgc tgcagatttg 1200 gatgatttct ccaaacaatt gcaacaatcc atgagcagtg ctgactcaac tcaggcctaa 1260                                                                         1260

Claims (8)

Primer set for reverse transcriptional loop-mediated isothermal amplification to detect 2019 new Coronavirus, which includes the oligonucleotide primers of SEQ ID NOs: 1-6. A composition for detecting 2019 new Coronavirus (2019 novel Coronavirus) comprising the primer set for reverse transcription ring isothermal amplification according to claim 1. A kit for detecting 2019 new coronavirus (2019 novel Coronavirus), comprising a primer set for reverse transcription ring-mediated isothermal amplification according to claim 1 and a reagent for performing ring-mediated isothermal amplification. According to claim 3, The kit is characterized in that it further comprises a lysis buffer consisting of 30 ~ 500 mM Tris of pH 6.0 ~ 7.0, containing 0.1 ~ 1.5% (v / v) of Triton X-100 2019 new coronavirus detection kit. Separating total RNA from biological samples isolated from patients suspected of 2019 new coronavirus infection;
Amplifying the target sequence by using the isolated total RNA as a template, and using the primer set for reverse transcription loop isothermal amplification of claim 1 to reverse reverse transcriptional loop-mediated isothermal amplification; And
The step of detecting the product of the amplification step; 2019 method for detecting a new coronavirus (2019 novel Coronavirus). Pre-processing biological samples separated from suspected patients with 2019 new coronavirus infection with lysis buffer;
Amplifying a target sequence by a reverse transcriptional loop-mediated isothermal amplification reaction using the sample pre-treated with the lysis buffer using the primer set for reverse transcription loop isothermal amplification of claim 1; And
The step of detecting the product of the amplification step; 2019 method for detecting a new coronavirus (2019 novel Coronavirus). According to claim 6, The lysis buffer containing 0.1 ~ 1.5% (v / v) of Triton X-100, 2019 new coronavirus (2019), characterized in that consisting of 30-500 mM Tris of pH 6.0-7.0 novel Coronavirus). The method according to claim 5 or 6, wherein the detection of the amplification product is carried out through fluorescence, turbidity, sediment generation by spindown, or electrophoresis.

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