KR102295574B1 - Human betacoronavirus universal primer sets for whole genome amplification method and diagnosis kit - Google Patents

Abstract

The present invention relates to a method for obtaining a whole genome sequence to determine whether beta-coronavirus infection is present and a use thereof. The method, a kit, and a composition of the present invention include a universal primer set capable of amplifying the genome sequence of the beta-coronavirus, and thus, it is possible to easily and quickly check the presence or absence of the target beta-coronavirus in a sample and related diseases. In addition, it can be effectively used to identify new beta-coronaviruses by determining whether or not the new beta-coronavirus is mutated through the whole genome analysis.

Description

Human betacoronavirus universal primer sets for whole genome amplification and a diagnostic kit using the same {Human betacoronavirus universal primer sets for whole genome amplification method and diagnosis kit}

The present invention relates to a technique for obtaining full genome information by amplifying the viral genome in a trace amount of a sample using a primer set capable of amplifying the full genome of human beta coronavirus and analyzing the nucleotide sequence.

Coronavirus (coronavirus) belongs to the coronaviridae (Coronaviridae) and is a single-stranded positive RNA virus with a size of about 100 to 120 nm having a spherical outer membrane, the outermost spike (S) protein, hemagglutinin-S It consists of five structural proteins: hemagglutinin-esterase (HE) protein, transmembrane (M) protein, small membrane (E) protein and nucleocapsid (N) protein. Coronavirus (Coronavirus) has a positive-sense single-stranded RNA genome of 30 kb, which is the largest among RNA viruses.

The coronavirus was first discovered in chickens in 1937, then in animals such as bats, cats, cattle, mice, dogs, pigs, and birds, and then in humans in 1965. HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 are known as common human coronaviruses that infect humans. Until now, coronavirus has been recognized as a pathogen that rarely infects humans and mainly infects animals such as dogs, pigs, and cattle. There were only cases where it caused intestinal diseases, such as diarrhea, which were not very dangerous for people. However, Severe Acute Respiratory Syndrome (SARS), which first appeared in mid-March 2003 and caused more than 100 deaths and more than 3,000 cases worldwide, and Severe Acute Respiratory Syndrome (SARS), which first occurred in Saudi Arabia in September 2012, caused 600 cases worldwide. As it became known that the causative agent of Middle East Respiratory Syndrome (MERS), which caused more than 1,980 deaths and 1,980 patients, was a novel (mutated) coronavirus, it began to draw attention.

Coronaviruses have four main subgroups known as alpha, beta, gamma, and delta. Among alpha corona viruses, there are two human-infectious species, 229E and NL63, and beta-coronaviruses are OC43, HKU1, and Middle East Respiratory (MERS-CoV). Syndrom coronavirus) and SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) are known, and are zoonotic viruses that can cause colds or severe pneumonia.

Human betacoronavirus OC43 (HCoV-OC43) is a betacoronavirus that infects humans and cattle. Infectious coronaviruses are enveloped positive-sense single-stranded RNA viruses that enter host cells by binding to the N-acetyl-9-O-acetyl neuraminic acid receptor.

Although the discovery of the human coronavirus was early, it received little attention due to its low medical significance and difficulties in isolation and identification, before being diagnosed with SARS in 2003. However, a sensitive and rapid detection method that can detect all kinds of human coronaviruses does not exist, and diagnosis has been made using monoclonal antibodies or PCR method.

Korean Patent No. 1857685 discloses primers and probes for simultaneously detecting subtypes of the genus coronavirus and a method for detecting coronavirus using the same, and Korean Patent No. 625325 encodes a SARS virus leader sequence or N protein. It discloses a probe composition derived from a nucleotide sequence and a primer and a method for detecting a coronavirus using the same.

However, a universal primer set for full genome detection of human beta coronavirus of the present invention has not yet been disclosed.

Korean Patent No. 1857685 Korean Patent No. 625325

Complete Genome Sequence of Human Coronavirus OC43 Isolated from Mexico. Genome Announc. 2016 Nov-Dec; 4(6): e01256-16. Characterization and Complete Genome Sequence of a Novel Coronavirus, Coronavirus HKU1, from Patients with Pneumonia. J Virol. 2005 Jan; 79(2): 884-895.

The present invention has been derived from the above needs, and the present inventors utilize a universal primer set that can be amplified in human beta coronavirus to quickly and conveniently sequencing the genome amplified through PCR, etc. from a trace amount of genome. The present invention was completed by confirming that the whole genome sequence could be secured.

In order to solve the above problems, the present invention provides a method for confirming the whole genome sequence of beta-coronavirus in an isolated sample comprising the following steps.

The sequencing method comprises the steps of (a) preparing one or more forward and reverse primer sets complementary to the whole genome of beta coronavirus;

(b) isolating RNA from the sample;

(c) performing a reverse transcription reaction using the isolated RNA;

(d) performing a polymerase chain reaction by adding a primer set to the sample on which the reverse transcription reaction has been performed; and

(e) analyzing the sequence of the amplified product.

In addition, the primer set is SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO:23 and SEQ ID NO:24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; and the primer sets of SEQ ID NO: 33 and SEQ ID NO: 34.

The present invention provides a beta-coronavirus diagnostic kit comprising one or more forward and reverse primer sets complementary to the full-length beta-coronavirus genome,

The primer set includes SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO:23 and SEQ ID NO:24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; And it provides a beta-coronavirus diagnostic kit comprising a primer set of SEQ ID NO: 33 and SEQ ID NO: 34.

The present invention provides a composition for diagnosing beta-coronavirus-induced diseases comprising one or more forward and reverse primer sets complementary to the full-length beta-coronavirus genome,

The primer set includes SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO:23 and SEQ ID NO:24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; And it provides a composition for diagnosing a beta-coronavirus-induced disease comprising a primer set of SEQ ID NO: 33 and SEQ ID NO: 34.

The features and advantages of the present invention are summarized as follows.

The present invention relates to a method for obtaining a sequence of the human beta coronavirus full-length genome and to a use thereof.

The method, kit and composition of the present invention use a set of forward and reverse primers capable of amplifying the whole genome of human beta coronavirus, and through genome amplification, a trace amount of virus samples and uncultured clinical samples of human beta coronavirus. It is easy and quick to check the presence or absence of a variant.

Accordingly, the methods, kits and compositions of the present invention enable accurate and rapid acquisition of the whole genome sequence of human beta coronavirus, and can be effectively applied to the diagnosis of diseases caused by human beta coronavirus.

1 shows the results of genome amplification of HCoV-OC43 using the primer set for beta coronavirus of Table 1 according to an embodiment of the present invention.
Figure 2 shows the results of the genome amplification of HCoV-OC43 using the primer set for beta corona virus of Table 2 or Table 3 according to an embodiment of the present invention.
Figure 3 shows the assembly result of beta corona virus whole genome Sanger sequencing according to an embodiment of the present invention.
4 shows a conceptual diagram of an exemplary flow of an optimal diagnostic protocol for beta-coronavirus in an embodiment of the present invention.
5 shows the quality analysis results for Miseq and Pacbio analysis of beta corona virus according to an embodiment of the present invention.
6 shows the quality analysis result of the NGS basic result according to an embodiment of the present invention. (A) Miseq. (B) Pacbio.
7 shows the quality analysis results of the Nanopore minION sequencing basic results according to an embodiment of the present invention.
Figure 8 shows the Nanopore minION real-time sequencing process for the beta-coronavirus genome according to an embodiment of the present invention.
9 shows a bioinformatic pipeline for whole genome analysis according to an embodiment of the present invention.
Figure 10 shows the Nanopore minION real-time sequencing process for the beta-coronavirus genome according to an embodiment of the present invention.
11 illustrates depth coverage of reference mapping according to an embodiment of the present invention. (A) Miseq. (B) Pacbio. (C) MinION.

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details such as specific components are shown, which are provided to help a more general understanding of the present invention, and it is common in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge of And, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted.

In order to achieve the object of the present invention, the present invention provides a method for confirming the whole genome sequence of beta coronavirus in an isolated sample comprising the following steps.

The sequencing method comprises the steps of (a) preparing one or more forward and reverse primer sets complementary to the whole genome of beta coronavirus; (b) isolating RNA from the sample; (c) performing a reverse transcription reaction using the isolated RNA; (d) performing a polymerase chain reaction by adding a primer set to the sample on which the reverse transcription reaction has been performed; and (e) analyzing the sequence of the amplified product.

In addition, the primer set is SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO:23 and SEQ ID NO:24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; and the primer sets of SEQ ID NO: 33 and SEQ ID NO: 34.

The concentrations of the primers are 0.1 to 1.0 pM, 0.1 to 0.8 pM, 0.1 to 0.6 pM, 0.2 to 1.0 pM, 0.2 to 0.8 pM, 0.2 to 0.6 pM, 0.3 to 1.0 pM, 0.3 to 0.8 pM, 0.3 to 0.6 pM, respectively. , 0.4 to 1.0 pM or 0.4 to 0.8 pM, for example, may be 0.4 to 0.6 pM, but is not limited thereto.

The step of carrying out the polymerase chain reaction is 30 to 50 cycles, 30 to 46 cycles, 30 to 44 cycles, 33 to 50 cycles, 33 to 46 cycles, 33 to 44 cycles, 36 to 50 cycles, 36 to 46 cycles, 36 to 44 cycles, 38 to 50 cycles or 38 to 46 cycles, for example, may be performed under 38 to 44 cycle conditions, but is not limited thereto.

The sample may be isolated from blood, serum, sputum, urine, or living tissue, but is not limited thereto.

The beta coronavirus may include one or more selected from the group consisting of OC43 and HKU1, but is not limited thereto.

The primer set may additionally include one or more primers selected from SEQ ID NOs: 35 to 199 or a reverse complementary primer sequence thereof.

In the step of analyzing the sequence of the amplified product, any sequencing method known in the art may be used regardless of the type, and preferably the Sanger method or next generation sequencing (NGS) may be used, but limited thereto it's not going to be

In order to identify chromosomal abnormalities including DNA copy number mutations (CNVs), which appear due to a deficiency or overlap of a part of a chromosome, various tests such as karyotyping, fluorescence orthosynthesis, chromosome microarray, and NGS-based screening are being performed ( Capalbo A, et al. 2017, Hum Reprod. Vol. 32(3), pp. 492-498). The karyotyping analysis has a lower resolution of about 5 Mb compared to other tests, and it is impossible to detect chromosomal deletions/duplications smaller than that. Chromosomal deletions and duplications of less than 5 Mb are called microdeletions/duplications, and the ratio of microdeletions/duplications among diseases caused by a single gene corresponds to 15% of all mutations (Vissers LE, et al. 2005, Hum). Mol Genet. Vol. 15;14 Spec No. 2:R215-23.).

In order to detect these microdeletions/duplications, fluorescence in situ hybridization (FISH) using a probe complementary to a specific nucleotide sequence and chromosomal microarray testing are being conducted. Fluorescence in situ hybridization is a test method that checks whether a specific nucleotide sequence exists in a chromosome by attaching a fluorescent label to a probe complementary to the nucleotide sequence to be confirmed. Since it shows a resolution of 100kb-1Mb, it is possible to detect microdeletions/duplications, but there is a disadvantage in that only known mutations can be detected because only the parts complementary to the probe sequence can be identified.

Currently, microarray-based comparative genomic hybridization (aCGH) is being used as the most common test method to check chromosomal microdeletion/duplication (Russo CD, et al. 2014, Cancer Discov. Vol. 4(1), pp. 19-21). The size of the CNV detectable through the microarray is determined by the density of the probe, and it is possible to detect CNVs with a size of approximately 50 kb. (Watson CT, et al. 2014) However, chromosomal abnormalities caused by chromosomal rearrangements such as translocation or inversion cannot be detected.

The technology for determining the nucleotide sequence sequence of the genes constituting living organisms is divided into the first-generation Sanger method and the next-generation sequencing method, NGS (Next Generation Sequencing). The first-generation method called Sanger sequencing, developed around 1977, is a chain-stop in which ddNTPs (di-deoxynucleotide triphosphates) stop the synthesis of DNA strands during the Polymerase Chain Reaction (PCR) process. It is a method to collect amplified DNA fragments by applying the chain-termination principle to confirm the nucleotide sequence. Although this method has high accuracy, it takes a long time to determine the nucleotide sequence and has problems in that high cost occurs.

In order to overcome these problems, a next-generation sequencing method has been developed. After segmenting the genetic information of numerous genomes, amplifying them to obtain big data, and then using bioinformatics to secure whole genome sequences (WGS) of the desired organism. Next-generation sequencing (NGS) is a sequencing method that divides chromosomes into small pieces and analyzes the genetic information of each piece in parallel. With the development of genetic analysis technology, NGS is being used as a screening test for genetic diseases in newborns because of its relatively short test time and cost, and its high resolution capable of detecting single nucleotide polymorphisms (SNPs) and indels (INDELs). However, due to the principle nature of NGS, which divides and analyzes chromosomes into small ones, there are technical limitations in detecting structural variations or CNVs of large-scale chromosomes (Yohe S, Thyagarajan B. 2017, Arch Pathol Lab Med. Vol. 141(11), pp. 1544-1557).

However, NGS is capable of detecting chromosomal abnormalities caused by chromosomal rearrangements that cannot be detected in probe-based microarrays and previously unknown new CNVs (Talkowski ME, et al. 2011, Am J Hum Genet. Vol. 88). (4), pp. 469-81). In addition, it has the advantage of showing higher coverage and resolution than microarrays and detecting breakpoints where chromosomal abnormalities start due to the characteristic of sequencing the chromosomes by fragmenting them into small pieces (Zhao M, et al. 2013, BMC). Bioinformatics. Vol. 14, Suppl 11:S1).

In order to achieve the object of the present invention, the present invention provides a beta-coronavirus diagnostic kit comprising one or more forward and reverse primer sets complementary to the beta-coronavirus full-length genome,

The primer set includes SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO:23 and SEQ ID NO:24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; And it provides a beta-coronavirus diagnostic kit comprising a primer set of SEQ ID NO: 33 and SEQ ID NO: 34.

In addition, the present invention is a composition for diagnosing beta-coronavirus-induced disease comprising one or more forward and reverse primer sets complementary to the full-length beta-coronavirus genome,

The primer set includes SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO:23 and SEQ ID NO:24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; And it provides a composition for diagnosing a beta-coronavirus-induced disease comprising a primer set of SEQ ID NO: 33 and SEQ ID NO: 34.

In this case, the beta-coronavirus-induced disease may be a respiratory disease, and the respiratory disease may include, but is not limited to, flu, cold, sore throat, bronchitis or pneumonia.

Advantages and features of the present invention, and methods for achieving them, will become apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the technical field to which the present invention belongs It is provided to fully inform the possessor of the scope of the invention, and the present invention is only defined by the scope of the claims.

[Example]

Example 1. Beta coronavirus universal primer set selection

At the end of March 2019, RNA of HCoV-OC43, a beta-coronavirus, was provided and secured from the Korea Research Institute of Chemical Technology. The concentration of RNA was 7.6 ng/μl in one tube, and A260/A280 was 8.98. A screening target primer was prepared by referring to the primer selection criteria among a total of 160 candidate groups for beta-coronavirus universal primers designed by the Korea Research Institute of Chemical Technology. Selection criteria were 1) the length of the amplification product is about 3 kb, 2) no dimer is formed, 3) 3 or less degeneracy, 4) T _m = about 55°C primer pair was selected, 5) nucleotide sequence In order to prevent sequence omission during analysis, it was considered that each amplification product overlapped by about 100 bp or more.

First strand cDNA synthesis was performed using the random hexamer method. Invitrogen's Superscript III First-Strand Synthesis SuperMix (Cat No. 18080-400) was used for first strand cDNA synthesis, and the method was performed according to the kit's manual. In this case, the RNA concentration was 7.6ng/ul. PCR was performed using a primer candidate using cDNA as a template. PCR mixture was prepared by putting 2μl first strand cDNA, 1μl forward primer, 1μl reverse primer, 10μl KAPA hotstart ready mix, and 6μl sterile distilled water into 0.2μl PCR tube and mixing. During the screening process, the amplification efficiency using the beta-coronavirus primer set was judged to be good, and the PCR conditions were changed as follows to shorten the construction time of the genome library. Through this, the PCR time could be reduced from 150 minutes to 80 minutes.

Conventional method (takes 150 minutes):

① 94℃ 5 min, ② [denaturation 94℃ 30 sec, annealing 55℃ 1 min, extension 72℃ 3 min] × 30 times, ③ final extension 72℃ 10 min.

How to change (takes 80 minutes):

① 98℃ 30sec, ② [denaturation 98℃ 5sec, annealing 55℃ 30sec, extension 72℃ 2min] × 30 times, ③ final extension 72℃ 2min.

As a result of screening for candidate primers under the above conditions, the primers in Table 1 below were preferentially selected as a set for beta corona.

<Priority selection primer set for beta-coronavirus whole genome amplification>

Forward Reverse Size (bp) One 89F 3039R 2950 2 - 1 1347F 4918R 3571 2 - 2 1354F 4918R 3564 3 4384F 7486R 3102 4 6448F 9698R 3250 5 8886F 11778R 2892 6 11551F 14637R 3086 7 14218F 17217R 2999 8 16674F 19230R 2556 9 18885F 21252R 2367 10 - 1 20419F 24290R 3871 10 - 2 21118F 24290R 3172 11 22461F 25820R 3359 12 24284F 26998R 2714 13 - 1 26998F 30476R 3478 13 - 2 27079F 30476R 3397

As a result of PCR for the primers in Table 1, it was confirmed that the set was synthesized up to the poly A tail, the last part of the OC43 genome, and the entire genome was covered (FIG. 1). However, it was confirmed that primer 2 forms a multiband, and a total screening was performed on other candidate primers to replace it. decided to do In addition, since it was determined that the 10th and 13th primer set sections had lower synthesis efficiency than other sections, two primers were selected for this section to ensure versatility.

Example 2. Complementary and final selection of beta-coronavirus universal primer set

As for the primer sequences in Table 1, it was decided to supplement the primer set of the corresponding region in order to improve the sequencing efficiency by supplementing the regions with low read coverage or unstable regions during the sequence assembly process for each primer, thereby designing a new primer. In the PCR process of the designed primer set, the instability of amplification efficiency was raised in the electrophersis result of the 10th primer amplicon. In addition, since there was not enough overlap between amplicon 12 and amplicon 13, there was a possibility that the genome assembly would not proceed properly when analyzing the genome of beta corona virus later. was produced.

Betacoronavirus primer set complementation experiments were performed. Based on the primers first selected in the third year, candidate primers that can be replaced and supplemented in the 10th and 12th amplicon regions were selected and PCR was performed in the same manner. A primer with more certain amplification efficiency of the 10th part was selected, and a new primer was selected and supplemented to increase the coverage efficiency of the 12th and 13th parts (FIG. 2, Table 2). In addition, the number of different cases was prepared by selecting the primer sets of regions 2, 10, and 14 as 2x coverage. It was decided to confirm the sequence using the replaced and supplemented primers. After that, after confirmation and complete 1x coverage, it was decided to conduct whole genome analysis using various next-generation sequencing methods (NGS).

<Final primer set for beta-coronavirus whole genome amplification>

Forward Reverse Size (bp) One 89F
(SEQ ID NO: 1) 3039R
(SEQ ID NO: 2) 2950 2 - 1 1347F
(SEQ ID NO: 3) 4918R
(SEQ ID NO: 4) 3571 2 - 2 1354F
(SEQ ID NO: 5) 4918R
(SEQ ID NO: 6) 3564 3 4384F
(SEQ ID NO: 7) 7486R
(SEQ ID NO: 8) 3102 4 6448F
(SEQ ID NO: 9) 9698R
(SEQ ID NO: 10) 3250 5 8886F
(SEQ ID NO: 11) 11778R
(SEQ ID NO: 12) 2892 6 11551F
(SEQ ID NO: 13) 14637R
(SEQ ID NO: 14) 3086 7 14218F
(SEQ ID NO: 15) 17217R
(SEQ ID NO: 16) 2999 8 16674F
(SEQ ID NO: 17) 19230R
(SEQ ID NO: 18) 2556 9 18885F
(SEQ ID NO: 19) 21252R
(SEQ ID NO: 20) 2367 10 - 1 20419F
(SEQ ID NO: 21) 24290R
(SEQ ID NO: 22) 3871 10 - 3 20419F
(SEQ ID NO: 23) 23207R
(SEQ ID NO: 24) 2788 11 22461F
(SEQ ID NO: 25) 25820R
(SEQ ID NO: 26) 3359 12 24290F
(SEQ ID NO: 27) 27544R
(SEQ ID NO: 28) 3254 13 24284F
(SEQ ID NO: 29) 26998R
(SEQ ID NO: 30) 2714 14 - 1 26998F
(SEQ ID NO: 31) 30476R
(SEQ ID NO: 32) 3478 14 - 2 27079F
(SEQ ID NO: 33) 30476R
(SEQ ID NO: 34) 3397

Example 3. Beta coronavirus universal primer set validation

After reverse transcription, PCR amplification was performed with the finally selected 14 primer sets, and the amplification efficiency was confirmed through electrophoresis (FIG. 2). In addition, the effectiveness of the primers was confirmed by the Sanger sequencing primer walking method for 14 amplification products of beta coronavirus. To confirm the accuracy of the single band of the amplification product of the selected primer, PCR purification was performed and the sequence of all genome parts was confirmed. In Sanger sequencing, as a result of combining 59 primer walking sequences, the entire genome of beta coronavirus was covered, and most of the nucleotide sequences were consistent (FIG. 3).

Example 4. Beta coronavirus whole genome analysis

The beta-coronavirus optimal diagnostic protocol was developed, and using the final beta-coronavirus primer set in Table 2 previously selected, several steps of preprocessing for whole genome analysis were performed as shown in FIG. 4 and established. Confirmation of the possibility of NGS sequencing for whole genome analysis of beta-coronavirus through preprocessing as shown in FIG. 4 was performed in three different ways using Illumina Miseq, Pacific Bioscience RSII, and OxfordNanopore MinIon. The amplification products produced using the final selected universal primer were mixed at the same concentration and sequenced according to the quality and quantity of each sequencing method. An optimal preprocessing and data analysis workflow for each NGS sequencing method was developed, and sequencing analysis and sequencing method were performed using this.

○ Beta coronavirus whole genome sequencing

1) NGS sequencing library preparation

- Illumina Miseq sequencing: The necessary conditions for the Truseq method of Illumina Miseq sequencing were concentration (ng/ul) > 10, Total Amount (ug): 0.4, and A260/280 Ratio >1.8. Therefore, for library preparation, a generous total of 0.579ug sample was prepared and prep was performed in the 301 paired-end method (FIG. 5). Figure 5 (B) is a Miseq amplicons, Figure 5 (D) is a Miseq Library quality analysis results.

- PacBio RS II sequencing: The necessary conditions for the PacBio RS II 10kb Amplicon Target sequencing method were concentration (ng/ul) > 150, Total Amount (ug): 10, A260/280 Ratio >1.8. However, to proceed in the amplicon method, the total amount of 5.46ug was sufficient for library preparation, so the sample was prepared in the corresponding amount (FIG. 5). Figure 5 (A) is Pacbio amplicons, Figure 5 (C) is the Pacbio Library quality analysis results.

- Nanopore MinION sequencing: The library preparation conditions for using the Oxford Nanopore MinION sequencing FLO-MIN106 flowcell and 1d amplicon/cDNA by Ligation kit were Total Amont (ug): 1-1.5, A260/280 Ratio >1.8. Therefore, the purified amplification products of beta-coronavirus were adjusted to the same concentration and mixed to give a total amount of about 2ug, and used to prepare the MinION sequencing library, and the library was prepared according to the standard protocol.

2) NGS sequencing raw data quality check control

- Illumina Miseq sequencing: For raw data sequenced by the Illumina Miseq 301 Paired-end method, two files of about 500Mb each were produced, producing a total of 1Gb raw data in fastq format. For quality check control of raw data, fastqc program was used to analyze raw data in fastq format. As a result, since most Q-scores are 30 or higher, quality can be measured (FIG. 6A).

- PacBio RS II sequencing: Sequencing was performed using the PacBio RS II Amplicon Target sequencing method, and about 1 Gb of raw data was produced. As a result of the raw data, 2,201,460,493bp were produced (FIG. 6B).

- Nanopore MinION sequencing: Nanopore sequencing using 1D ligation method resulted in sequencing for about 21 hours, resulting in 44.1 Gb of raw data in fast5 format. After converting this to fastq format using Guppy basecaller V.2.3.1, quality check control was performed. As a result, data of 2.96 Gb was generated. When the quality check of the entire base was performed using OxfordNanopore's EPI2ME - Fastq control experiment (V3.2.2) program, the average Q-score was 9.01, but the target length confirmed by the fastqc program. The Q-score value of was increased to an average of about 14 (Fig. 7).

Real-time confirmation of NGS sequencing of beta-coronavirus is possible only with MinION, so the Oxford Nanopore MinION sequencing real-time progress was checked to determine whether real-time sequencing is possible. Total running time was about 21h. After nanopore sequencing running, a total of 455.389 reads (about 44.1GB) raw data were calculated, and the throughput read length was about 3 kb ( FIG. 8 right panel). 44.1GB of fast5 format raw data calculated by MinION equipment was converted into fastq file using OxfordNanopore's own program, Guppy software. This file conversion took a total of 10h to correct the basecalling and sequence quality.

Example 5. Beta coronavirus whole genome analysis and nucleotide sequence determination

○ Beta-CoV whole genome sequencing analysis and sequencing method

- Illumina Miseq sequencing: A total of 1 Gb of Illumina Miseq raw data sequenced with 301 Paired-End was QC and trimmed using CLC genomic workbench, and then merged. Two types of long contigs were obtained by mapping and de novo assembly to the reference genome using the merged and extended data.

- PacBio RS II sequencing: After converting 23.8G of Pacbio raw data into bam file format, a consensus sequence was obtained using SMRTlink's Long Amplicon Analysis (LAA), a Pacbio exclusive analysis program. The obtained sequences were assembled using the Megahit program to obtain a final long contig. In addition, by mapping the fastq format generated from rawdata to the reference genome using the CLC genomic workbench program, a consensus sequence was generated to generate two versions of the final contig.

- Nanopore MinION sequencing: As a result of sequencing for about 21 hours, MinION raw data of 44.1G (fast5 format) was calculated, and raw data was handled in two ways. First, it was converted into a 2.96Gb fastq format file using Guppy software (V2.3.1) using the HMM method. This file conversion took about 10h with the fast option setting as the basecalling and sequence quality correction process. The complete genome sequence consisting of one long contig consensus sequence was obtained by mapping the file converted to fastq to the reference genome using CLC genomic workbench. Next, after making MinION rawdata into one file, alignment was done using trim and reference mapping using bowtie2 program. The analyzed BAM file was subjected to Variant Discovery using Genome Analysis Tool Kit (GATK) to derive a vcf file and converted to the FASTA file format to obtain one contig.

- Sanger Sequencing: All 14 amplicons were sequenced using the Primer walking method. All of the obtained nucleotide sequences were checked for quality by referring to the chromatogram for bases above Q20, and trimmed using the ezeditor2 program. To evaluate the accuracy of each sequencing method, a hybrid assembly was performed using a codoncode alinger with each contig derived from Pacbio, MinION, and Miseq sequencing.

○ Results of beta-coronavirus whole genome sequencing: As a result of sequencing using four different methods, it was confirmed that one final consensus sequence was generated. The nucleotide sequence obtained in this way was compared with the reported beta coronavirus whole genome (GenBank Accession number AY391777.1) and the nucleotide sequence (Fig. 10, Table 3, Table 4). In this study, based on the results of Miseq, Pacbio, MinIon, and Sanger sequencing, one final contig was obtained and compared primarily by reference mapping according to each sequencing analysis method. In addition, similar final contigs were obtained when de novo assembly was performed using the Miseq and PacBio methods, which are known to have high accuracy among the NGS sequencing methods reported so far. A consensus sequence was created using these two methods and compared with the reference. The depth coverage of the reference mapping was different for each sequencing method as shown in FIG. 11 . Consensus and reference confirmed that about 66 nts were inconsistent. However, when compared with the consensus sequence for each final contig sequence according to the sequencing method, about 9 or less sequences were mismatched. This means that there is a variation in the level of microevolution in the experimental strain compared to the reference strain. Compared with the consensus sequence, MinIon has 3, Pacbio 5, Miseq 4, and Sanger 4 sequences inconsistent. The error rate is PacBio, and as a result of reference mapping, Miseq sequencing has high coverage and low error rate.

<Comparison of beta-coronavirus genome analysis performance of four sequencing technologies>

<Sequence comparison of beta-coronavirus whole genome obtained by Miseq, PacBio, MinIon, and Sanger sequencing>

<110> KRISS <120> Human betacoronavirus universal primer sets for whole genome amplification method and diagnosis kit <130> table4 <160> 199 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta89F <400> 1 gattgyggtt ttgttatgca gt 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta3039R <400> 2 tcyccagcct catcaaataa a 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta1347F <400> 3 gattgyggtt ttgttatgca gt 22 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta4918R <400> 4 aaattaacac catcwacagt 20 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Beta1354F <400> 5 ggttttgtta tgcagtwtgg tta 23 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta4918R <400> 6 aaattaacac catcwacagt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta4384F <400> 7 gctggtatat ttagtgtkcc 20 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Beta7486R <400> 8 ccagtrccac cattagc 17 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta6448F <400> 9 acaggttgtr aktatgttgt t 21 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta9698R <400> 10 gtaatcataa aagtagtrag ag 22 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta8886F <400> 11 tttttatgct agtggykgtg t 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta11778R <400> 12 tagaattaga agcwacatgc 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta11551F <400> 13 tttagaatgc ctwtgggtgt t 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta14637R <400> 14 agtaatwgca gcattaccat c 21 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta14218F <400> 15 ggtgtattat wcattgtgct aa 22 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta17217R <400> 16 gtaagcatrc taacttcatc 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta16674F <400> 17 aagacagttt taggtgagta t 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta19230R <400> 18 cacacaaggm gtatctgaat a 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta18885F <400> 19 cttaaagctg ccatgctmtg 20 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta21252R <400> 20 atgcataaca ttkccatcta t 21 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta20419F <400> 21 gtagtaagag tgtttgyac 19 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta24290R <400> 22 ccacsttstt gataaaaatg aa 22 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta20419F <400> 23 gtagtaagag tgtttgyac 19 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta23207R <400> 24 gaatcaacaa cctgyacrgg 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta22461F <400> 25 ttatttggtg ayagtcgttc 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta25820R <400> 26 aacrcaacca agataactat 20 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta24290F <400> 27 ttcattttta tcaasaasgt gg 22 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta27544R <400> 28 taccaaggcc atttnacata 20 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Beta24284F <400> 29 tgtattttca tttttatcaa gaa 23 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta26998R <400> 30 acaaytcat taaccttctc 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta26998F <400> 31 gagaaggtta atgartgtgt 20 <210> 32 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Beta30476R <400> 32 gcgaggggtw accacc 16 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta27079F01 <400> 33 cagaatgctc catatggttt g 21 <210> 34 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Beta30476R <400> 34 gcgaggggtw accacc 16 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta27079F02 <400> 35 aaaatgctcc ttatggtttg 20 <210> 36 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Beta30476R <400> 36 gcgaggggtw accacc 16 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta39F01 <400> 37 tgatctcttg tgatcttt tt 22 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta39F02 <400> 38 cgatctcttg tcagatcyca tt 22 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta75F01 <400> 39 ataaaaacat ccactccctg t 21 <210> 40 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta75F02 <400> 40 ttaaacaaga ttccctgtta tc 22 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta89F <400> 41 tccctgtwat cyatgcttgt g 21 <210> 42 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta165F01 <400> 42 gccagggacg tgttgtatc 19 <210> 43 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta165F02 <400> 43 gycagtgacg tgttggttg 19 <210> 44 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Beta194F <400> 44 cccncccata ggtcrca 17 <210> 45 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta221F <400> 45 aycarcaaat acggtctcg 19 <210> 46 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Beta248F01 <400> 46 tgggctccag aatttcca 18 <210> 47 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Beta248F02 <400> 47 tgggcgccag aatttcgt 18 <210> 48 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta382F01 <400> 48 tgtgatggtg gattgtcgc 19 <210> 49 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta382F02 <400> 49 tgtgaagata gattgtcgc 19 <210> 50 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta724F01 <400> 50 tacccttagc ytttatgcc 19 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta724F02 <400> 51 taccttttgg ttttatgcca t 21 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta885F <400> 52 catgmtgagc ctaarggtaa 20 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta965F <400> 53 tgtagaccag tatggytgtg a 21 <210> 54 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta1347F <400> 54 gattgyggtt ttgttatgca gt 22 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta1924F01 <400> 55 tttactgttt gtgcagatgg 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta1924F02 <400> 56 tttgcagttt gtggagatgg 20 <210> 57 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta3039F <400> 57 tttatttgat gaggctggyg a 21 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta3586F <400> 58 caaggtggtt wtgtwgctga 20 <210> 59 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta3699F01 <400> 59 ggatgctatg ttcttttatg g 21 <210> 60 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta3699F02 <400> 60 ggatgctktg tttttttatg g 21 <210> 61 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Beta4009F <400> 61 caattgtatg gttyktgtat aac 23 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Beta4033F <400> 62 cctaatgtrt gttttgttaa agg 23 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta4131F <400> 63 tggtggwgtt gcaaaagcta 20 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta4225F <400> 64 ggagattgtt atgtytctac 20 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta4384F <400> 65 gctggtatat ttagtgtkcc 20 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta4918F <400> 66 actgtwgatg gtgttaattt 20 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta5113F01 <400> 67 tttgatcaga aggaattgct 20 <210> 68 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta5113F02 <400> 68 tttgatcagc aacaattgct 20 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta5500F <400> 69 gttatgcatt ttggtacatt 20 <210> 70 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta6448F <400> 70 acaggttgtr aktatgttgt t 21 <210> 71 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta7285F <400> 71 tagctaatat gttaccwgc 19 <210> 72 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Beta7486F <400> 72 gctaatggtg gyactgg 17 <210> 73 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta7714F <400> 73 gatgatgttr atgctagttt 20 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta7821F <400> 74 aggyaatttt ctgaatgctg 20 <210> 75 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta7938F <400> 75 gtttgatgtt tatgtkgata c 21 <210> 76 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta8227F <400> 76 gcwgatttag gtgttctkat 20 <210> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta8266F <400> 77 gtrcagggta aygttgctaa 20 <210> 78 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta8599F <400> 78 gataatggtg ttrttagaga t 21 <210> 79 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta8662F <400> 79 tttratcaat ggtatgagtc 20 <210> 80 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta8886F <400> 80 tttttatgct agtggykgtg t 21 <210> 81 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta9220F <400> 81 tttgtggtag agatnttttt ga 22 <210> 82 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta9698F <400> 82 ctctyactac ttttatgatt ac 22 <210> 83 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta10279F <400> 83 acttttactg tyttagctgc 20 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta10333F <400> 84 acyatgcgta gtagttatac 20 <210> 85 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta10451F <400> 85 gtactggttg tcayactgg 19 <210> 86 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta10630F <400> 86 gaagattta atgtktgggc t 21 <210> 87 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Beta10735F <400> 87 ttggctgcta tyaagcgt 18 <210> 88 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Beta10909F <400> 88 acatttttgt ttagttgyat aatt 24 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta10936F <400> 89 gcatttgtka aatggactat 20 <210> 90 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta11040F <400> 90 taagcatytt tatttgacta tg 22 <210> 91 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta11551F <400> 91 tttagaatgc ctwtgggtgt t 21 <210> 92 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta11728F <400> 92 actgatgtsa aatgtgctaa tg 22 <210> 93 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta11776F <400> 93 ttgcatgtwg cttctaattc ta 22 <210> 94 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta11778F <400> 94 gcatgtwgct tctaattcta 20 <210> 95 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta11785F <400> 95 gcttctaatt ctargttgtg g 21 <210> 96 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Beta12649F <400> 96 cctactcaat gttaytataa taat 24 <210> 97 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta12703F <400> 97 cttagtgatg ttgatggtct t 21 <210> 98 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Beta12863F <400> 98 gaggstgggt tgttggta 18 <210> 99 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Beta13048F <400> 99 catgctggta cyggtatg 18 <210> 100 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Beta13051F <400> 100 gctggtacyg gtatggc 17 <210> 101 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta13470F <400> 101 aattgttgcc gttttcagcg t 21 <210> 102 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta13603F <400> 102 tggctgaaca ygatttcttt ac 22 <210> 103 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Beta13638F <400> 103 ggtagtcgyg tgccaca 17 <210> 104 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta13682F <400> 104 tactatgttr gatctttgct a 21 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta13692F <400> 105 gatctttgct atgcattgcg 20 <210> 106 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta13797F <400> 106 gattggtatg attttgttga a 21 <210> 107 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta13801F <400> 107 ggtatgattt tgttgaaaat cc 22 <210> 108 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta13960F <400> 108 aatggtatga ttttggtgay t 21 <210> 109 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Beta14109F <400> 109 gatcttgtrc agtatgattt tac 23 <210> 110 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Beta14145F <400> 110 garttgttta ataagtattt taag 24 <210> 111 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta14218F <400> 111 ggtgtattat wcattgtgct aa 22 <210> 112 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta14637F <400> 112 gatggtaatg ctgcwattac t 21 <210> 113 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta14751F <400> 113 tatgatggtg gktgtatac 19 <210> 114 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta14764F <400> 114 gtataccrgc atcacaagt 19 <210> 115 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta14772F <400> 115 gcatcacaag tyattgttaa t 21 <210> 116 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Beta14847F <400> 116 tattatgarg cattatcatt tga 23 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta15027F <400> 117 tgtttgaara gtatagcagc 20 <210> 118 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta15054F <400> 118 ggtgttcctg twgttatag 19 <210> 119 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta15082F <400> 119 aattttatgg tggytggga 19 <210> 120 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta15144F <400> 120 cttatgggyt gggattatcc t 21 <210> 121 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta15261F <400> 121 tatcgmcttg cgaatgaatg 20 <210> 122 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta15293F <400> 122 tgagtgaaat wgttatgtgt gg 22 <210> 123 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta15321F <400> 123 tattatgtta agcctggtgg 20 <210> 124 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta15351F <400> 124 gtgatgcaac yactgctttt g 21 <210> 125 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta15435F <400> 125 aatggcmata agattgaaga 20 <210> 126 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta15569F <400> 126 gatgattttg agtgatgatg g 21 <210> 127 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta15690F <400> 127 gaatcyaaat gttgggttga a 21 <210> 128 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta15817F <400> 128 taggagctgg wtgttttgt 19 <210> 129 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Beta15918F <400> 129 catgaaaatg aagaatacca aaa 23 <210> 130 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta16020F <400> 130 attttaagta cttgtgatgg 20 <210> 131 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta16213F <400> 131 atcataaata tgtyttgagt gt 22 <210> 132 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta16425F <400> 132 atagctagtt gtaaatggac 20 <210> 133 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta16674F <400> 133 aagacagttt taggtgagta t 21 <210> 134 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta16908F <400> 134 cacatggwa tgaaacgtta 20 <210> 135 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta17040F <400> 135 gaygcattgt gtgaaaaagc 20 <210> 136 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta17217F <400> 136 gatgaagtta gyatgcttac 20 <210> 137 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta17697F <400> 137 gattctgctc aaggttckga a 21 <210> 138 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Beta17745F <400> 138 gcagaaacag cscattc 17 <210> 139 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Beta18321F <400> 139 aaagctcctc ctggtga 17 <210> 140 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Beta18579F <400> 140 ggttgttggc gccatagt 18 <210> 141 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta18885F <400> 141 cttaaagctg ccatgctmtg 20 <210> 142 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta19029F <400> 142 ggtttrtgta tgttttggaa 20 <210> 143 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Beta19146F <400> 143 ttgtatgtta ataaacatgc att 23 <210> 144 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta19230F <400> 144 tattcagata ckccttgtgt g 21 <210> 145 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta19398F <400> 145 acagcaggyt ttactttttg 20 <210> 146 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta19816F <400> 146 tmctttttga tggtcgtga 19 <210> 147 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Beta20060F <400> 147 gagcccacaa ggtaatct 18 <210> 148 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta20419F <400> 148 gtagtaagag tgtttgyac 19 <210> 149 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta20715F <400> 149 tgtatgatga atgttgctaa gt 22 <210> 150 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta20760F <400> 150 aatactacaa cattagctgt 20 <210> 151 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta20869F <400> 151 ctggtastat tcttgtagat aa 22 <210> 152 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta20974F <400> 152 tgataatwtc tgatatgtat ga 22 <210> 153 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta21118F <400> 153 aaatgcttct tctagtgaag g 21 <210> 154 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta21252F <400> 154 atagatggma atgttatgca t 21 <210> 155 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta22461F <400> 155 ttatttggtg ayagtcgttc 20 <210> 156 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta24290F <400> 156 ttcattttta tcaasaasgt gg 22 <210> 157 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta25820F <400> 157 atagttatct tggttgygtt 20 <210> 158 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta26092F <400> 158 tctccyaaag ttactattga t 21 <210> 159 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta26932F <400> 159 aatgcttatg tytctcaaca 20 <210> 160 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta26998F <400> 160 gagaaggtta atgartgtgt 20 <210> 161 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta28774F <400> 161 atgtgtatwg atatgaaagg 20 <210> 162 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta29425F <400> 162 tcttttaaaa cagcygatgg 20 <210> 163 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Beta29623F <400> 163 actaggttty cgcctgg 17 <210> 164 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta29668F <400> 164 gaaggctcag gaaggtctg 19 <210> 165 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta30510F <400> 165 cactctctat cagaatgrat 20 <210> 166 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Beta1354F <400> 166 ggttttgtta tgcagtwtgg tta 23 <210> 167 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta1423F01 <400> 167 ggtaacatga tagatggttt t 21 <210> 168 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta1423F02 <400> 168 ggtaatatga tggatggttt t 21 <210> 169 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta4402F01 <400> 169 ccttctgatg tgtctttaac 20 <210> 170 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta4402F02 <400> 170 cctgctgatg tgtcattaac 20 <210> 171 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta4786F01 <400> 171 tttgagtgta ctggaggcat 20 <210> 172 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta4786F02 <400> 172 tttgagtgtc ctaattctat 20 <210> 173 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta22642F <400> 173 aattatyttt tatgagggtg tt 22 <210> 174 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Beta24284F <400> 174 tgtattttca tttttatcaa gaa 23 <210> 175 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta26826F01 <400> 175 atttggtgct ataagtkctt ct 22 <210> 176 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta26826F02 <400> 176 atttggtgcw attagttctt ct 22 <210> 177 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta27079F01 <400> 177 cagaatgctc catatggttt g 21 <210> 178 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta27079F02 <400> 178 aaaatgctcc ttatggtttg 20 <210> 179 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta27544F <400> 179 tatgtwaaat ggccttggta 20 <210> 180 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta28471F <400> 180 gaatggaatt tytctttggg t 21 <210> 181 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Beta28738F01 <400> 181 agtttttgga gtttyaaccc aga 23 <210> 182 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Beta28738F02 <400> 182 agttggtgga gttttaaycc aga 23 <210> 183 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Beta29134F01 <400> 183 agtagagcgt cctctgga 18 <210> 184 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Beta29134F02 <400> 184 agtagaagct cctctgga 18 <210> 185 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta29468F01 <400> 185 gatggtattt ttactatctg gg 22 <210> 186 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Beta29468F02 <400> 186 gatggtattt ctactatctc gg 22 <210> 187 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beta29684F <400> 187 ttgaaggctc aggaaggtc 19 <210> 188 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta29790F01 <400> 188 acctgacatg gctgatcaaa t 21 <210> 189 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta29790F02 <400> 189 acctgatatg gctgatgaga t 21 <210> 190 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Beta30046F01 <400> 190 gaactcgcac ccacagc 17 <210> 191 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta30046F02 <400> 191 gaattagctc ctacaccagg t 21 <210> 192 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Beta30476F <400> 192 ggtggtwacc cctcgc 16 <210> 193 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta30721F01 <400> 193 gccaattgga agaatcacaa 20 <210> 194 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta30721F02 <400> 194 gccttttgga ggaattacaa 20 <210> 195 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta22460F <400> 195 ttatttggtg ayagtcgttc 20 <210> 196 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta23207F <400> 196 ccygtrcagg ttgttgattc 20 <210> 197 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta23683F <400> 197 gctgttatag gwgatttwaa 20 <210> 198 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta24292F <400> 198 catttttatc aagaasgtgg 20 <210> 199 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta25819F <400> 199 gatagttatc ttggttgygt t 21

Claims (13)

Method for confirming the whole genome sequence of beta coronavirus in an isolated sample comprising the steps of:
(a) preparing one or more forward and reverse primer sets complementary to the beta coronavirus full-length genome;
(b) isolating RNA from the sample;
(c) performing a reverse transcription reaction using the isolated RNA;
(d) performing a polymerase chain reaction by adding a primer set to the sample on which the reverse transcription reaction has been performed; and
(e) analyzing the sequence of the amplified product;
The primer set includes SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO:23 and SEQ ID NO:24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; and the primer sets of SEQ ID NO: 33 and SEQ ID NO: 34. The method of claim 1, wherein the sample is isolated from blood, serum, sputum, urine, or living tissue. The method according to claim 1, wherein the beta coronavirus comprises one or more selected from the group consisting of OC43 and HKU1. The method according to claim 1, wherein the primer set further comprises one or more primers selected from SEQ ID NOs: 35 to 199 or a reverse complementary primer sequence thereof. A beta-coronavirus diagnostic kit comprising one or more forward and reverse primer sets complementary to the beta-coronavirus full-length genome,
The primer set includes SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO:23 and SEQ ID NO:24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; And a beta-coronavirus diagnostic kit comprising a primer set of SEQ ID NO: 33 and SEQ ID NO: 34. The kit according to claim 5, wherein the diagnostic sample is isolated from blood, serum, sputum, urine, or living tissue. The kit according to claim 5, wherein the beta coronavirus comprises one or more selected from the group consisting of OC43 and HKU1. The kit according to claim 5, wherein the primer set further comprises one or more primers selected from SEQ ID NOs: 35 to 199 or a reverse complementary primer sequence thereof. A composition for diagnosing a beta-coronavirus-induced disease comprising one or more forward and reverse primer sets complementary to the full-length beta-coronavirus genome,
The primer set includes SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO:23 and SEQ ID NO:24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; And SEQ ID NO: 33 and a composition for diagnosing a beta-coronavirus-induced disease comprising a primer set of SEQ ID NO: 34 The composition of claim 9, wherein the beta-coronavirus-induced disease is a respiratory disease. The composition of claim 9, wherein the diagnostic sample is isolated from blood, serum, sputum, urine, or living tissue. The composition of claim 9, wherein the beta-coronavirus comprises at least one selected from the group consisting of OC43 and HKU1. The composition of claim 9, wherein the primer set further comprises one or more primers selected from SEQ ID NOs: 35 to 199 or a reverse complementary primer sequence thereof.

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