WO2021091211A1 - Procédé de détection de génome entier utilisant l'amplification du génome entier d'un virus mers-cov, et kit de diagnostic - Google Patents

Procédé de détection de génome entier utilisant l'amplification du génome entier d'un virus mers-cov, et kit de diagnostic Download PDF

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WO2021091211A1
WO2021091211A1 PCT/KR2020/015281 KR2020015281W WO2021091211A1 WO 2021091211 A1 WO2021091211 A1 WO 2021091211A1 KR 2020015281 W KR2020015281 W KR 2020015281W WO 2021091211 A1 WO2021091211 A1 WO 2021091211A1
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mers
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sequence
corona virus
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김세일
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한국표준과학연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/101—Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention uses a primer set that can amplify the full-length genome of MERS corona virus (MERS-CoV), amplifies the viral genome from a small amount of samples, and analyzes the nucleotide sequence to analyze the full-length genome information. It relates to the technology to obtain exactly.
  • MERS-CoV MERS corona virus
  • Coronavirus has a 30 kb positive-sense single-stranded RNA genome, which is the largest among RNA viruses.
  • Beta-coronavirus a type of coronavirus, has 10 species, and a total of 13 complete genomes, including unclassified, are currently registered with NCBI.
  • MERS-CoV Middle East respiratory syndrome-related coronovirusm
  • group C of the beta corona virus is one of the complete genomes of the beta coronavirus.
  • Saudi Arabia, Jordan, Qatar, and the United Arab Emirates Kuwait, Turkey, Oman, Norway, Bangladesh, Austria, United Kingdom, South Korea, United States, China and Hong Kong.
  • MERS-CoV is the sixth coronavirus to be discovered similar to SARS. Initially, it was commonly used as '2012 new coronavirus', or'new coronavirus' for short, and until May 2013 when the World Health Organization accepted the name'MERS', it was also called'SARS-like virus', and'Middle East SARS', It was also called'Saudi SARS'. Based on the coronavirus sequence data from bats in South Africa, since the discovery of the Middle East Respiratory System (MERS-CoV, EMC-HCoV) in 2012, there have been 2229 confirmed cases of laboratory cases of MERS infection by June 2018, reported to the WHO. Became.
  • MERS-CoV Middle East Respiratory System
  • EMC-HCoV HCoV-EMC/2012
  • HCoV-EMC/2012 One strain of MERS-CoV, known as EMC-HCoV or HCoV-EMC/2012, discovered from the first patient in London, UK in 2012, was found to be 100% consistent with that from the Egyptian tomb bat (Taphozous perforatus), as above.
  • MERS disease has resulted in 791 deaths to date. Since May 2015, more than 100 infected people (186 people) have occurred in Korea, and 39 of the infected people have died, showing a mortality rate of about 21%. Typical symptoms of MERS are fever, cough, and short breathing. Pneumonia was common, and gastrointestinal symptoms were also reported. The majority of human infections of MERS are due to human-to-human infections, and the main host of MERS-CoV is dromedary camels, which are the animal causes of human infections. However, the exact role and method of the dromedary in the transmission or transmission of the virus is unknown.
  • the domestic epidemic ORF3 gene was about 4% different from the first isolate, but other genes were more than 99% similar. Appeared.
  • the E gene which induces viral particle formation and expresses the envelope protein, which is a component of the outer membrane, has no mutations, and is therefore presumed to play an important role in the transmission of viral infections.
  • some mutations, including amino acid mutation 529 of the RBD region, were identified in the S gene, but it was analyzed that it was difficult to determine the association between the pathogenicity of the virus or human infection only by analyzing the mutation at the gene level. Therefore, various biological characteristics analysis and research in experiments such as virus self-replication and infectivity analysis using susceptible cells, and pathogenicity analysis experiments using animal models for characterization of gene sites that are known to be important in viral infection transmission and pathogenicity. The need for development emerged.
  • the present inventors have developed a diagnostic kit and a full-length genome sequence method through amplification of the full-length MERS corona virus, and the method can accurately and quickly confirm the presence or absence of the MERS corona virus in a sample, and can detect mutations. ) Is confirmed to be 99.88%, and it can be effectively used for identification of new viruses by accurately discriminating genetic mutations in a specific, fast time.
  • the present invention using a general-purpose primer capable of amplifying MERS corona virus (MERS-CoV), amplified from a small amount of genome through PCR, etc., and quickly and accurately MERS through sequencing method.
  • MERS-CoV MERS corona virus
  • the present invention was completed by developing a method for securing the full-length genome sequence of the corona virus and a diagnostic method using the same.
  • An object of the present invention is to provide a method for confirming the full-length genome sequence of MERS coronavirus in a sample.
  • Another object of the present invention is to provide a diagnostic kit for MERS coronavirus in a sample.
  • Another object of the present invention is to provide a composition for detecting MERS coronavirus-induced diseases.
  • a primer set including two or more of SEQ ID NOs: 1 to 28 for detecting the MERS corona virus (Middle East respiratory syndrome-related corona virus, MERS-CoV) full-length genome.
  • MERS corona virus Middle East respiratory syndrome-related corona virus, MERS-CoV
  • the present invention provides a MERS corona virus diagnostic kit comprising a set of one or more forward and reverse primers complementary to the MERS corona virus full-length genome.
  • the present invention provides a composition for diagnosing MERS coronavirus-induced diseases comprising one or more forward and reverse primer sets complementary to the MERS coronavirus full-length genome.
  • a method for confirming the full-length genome sequence of MERS coronavirus in an isolated sample comprising the following steps is provided.
  • MERS-CoV Middle East respiratory syndrome-related corona virus
  • step (c) performing a reverse transcription reaction with the isolated RNA and the primer set of step (a);
  • the MERS-CoV includes human MERS coronavirus and bat MERS-like coronavirus.
  • the amplification of step (d) is a polymerase chain reaction, a nested-polymerase chain reaction, a multiplex polymerase chain reaction, and a competitive polymerase chain reaction.
  • Chain reaction real time polymerase chain reaction, cDNA chip, oligonucleotide chip, real time quantitative polymerase chain reaction, and loop-meditated isothermal amplification is one or more methods selected from the group consisting of,
  • Sequence analysis of the amplified product of step (e) is Sequel, MinION, MiSeq, GS FLX, Sanger Sequencing, SOLiD, Ion Torrent, HiSeq, BGISEQ sequencing, GeneReader NGS System, solid-state nanopore-based DNA sequencing, GenoCare This is one or more methods selected from the group consisting of Sequencer, GENIUS, Hyb & Seq sequencing, or iSeq 100.
  • the assembler of the MERS coronavirus full-length genome sequence is Quast, Bandage, CLC, LAA, Ezeditor, AbySS, IDMA-UD, MEGAHIT, metaSPAdes, Metavelvet, Omega, Ray Meta, SPAdes, IDBA, Meta-IDBA, MIRA. , SOAPdenovo, Velvet, Bowtie2, BMA, Yara and SMRT portal, Stampy, or one or more methods selected from the group consisting of MAQ, and extraction of nucleotide sequence mutation information of the full-length genome sequence of the MERS coronavirus (variant calling) is SOAPsnp, SAMtools , Picard, GATK is one or more methods selected from the group consisting of.
  • the diagnostic kit and full-length genome sequence confirmation method through MERS coronavirus full-length genome amplification provided by the present invention can quickly and easily check the presence of MERS coronavirus and genetic mutations in trace amounts of virus samples and uncultured clinical samples.
  • the method, kit, and composition of the present invention makes it possible to obtain the full-length genomic sequence of the MERS coronavirus very accurately and quickly, and through this, it can be effectively applied to the diagnosis of diseases caused by the human MERS coronavirus.
  • MP01 to MP07 are from Set01 to the selected primer set in Table 1 of the present invention. It means Set07, and MP07' to MP13 means Set08 to Set 14 in the selected primer set.
  • Figure 2 shows the results of the PCR (Electrophoresis) showing the MERS-CoV full-length genome amplification results of the selected primer set in Table 1 of the present invention.
  • Figure 3 shows the results of confirming the minimum amount of RNA for cDNA synthesis of the present invention.
  • Figure 4 shows the MERS-HCoV (human MERS corona virus) sequencing results diagnosed using the selected primers in Table 1 of the present invention.
  • the accuracy of the sequencing technology was compared based on the concensus sequence, and the sequence was confirmed by Sequel (Pacbio, Pacbio RSII, Pacbio), MinION (Oxford Nanopore Technology), and Miseq (Illumina).
  • Figure 6 shows the results of the bat MERS corona virus (btMERS or NeoCoV) genome assay results for confirming versatility
  • (A) is an overview of extension PCR and ligation experiments for the bat MERS corona virus (NeoCoV) genome fragment to confirm the versatility. Is shown.
  • (B) shows the position of the restriction enzyme for linking the genome segment of the bat MERS corona virus (NeoCoV) for confirming the versatility.
  • FIG 7 shows the result of electrophoresis (Electrophoresis) obtained by amplifying the full-length genome (PCR) of the bat MERS coronavirus (NeoCoV) genome for verification of the versatility of the present invention with the selected primer set of the present invention.
  • FIG. 10 shows a process for the MERS corona virus sequencing assemble method of the present invention.
  • step (c) performing a reverse transcription reaction with the isolated RNA and the primer set of step (a);
  • the sequence analysis of the amplified product of step (e) is Sequel, MinION, MiSeq, GS FLX, Sanger Sequencing, SOLiD, Ion Torrent, HiSeq, BGISEQ sequencing, GeneReader NGS System, solid-state nanopore-based DNA.
  • One or more methods selected from the group consisting of sequencing, GenoCare Sequencer, GENIUS, Hyb & Seq sequencing, or iSeq 100 may be used, and among the methods, Sequel (Pacbio, Pacbio RSII)), MinION (Oxford Nanopore Technology), The Miseq (Illumina company, Nanopore Min-ION) method is preferred.
  • the assembler of the MERS coronavirus full-length genome sequence is Quast, Bandage, CLC, LAA, Ezeditor, AbySS, IDMA-UD, MEGAHIT, metaSPAdes, Metavelvet, Omega, Ray Meta, SPAdes, IDBA, Meta- At least one method selected from the group consisting of IDBA, MIRA, SOAPdenovo, Velvet, Bowtie2, BMA, Yara and SMRT portal, Stampy or MAQ may be used, and LAA, Megahit, bowtie2, CLC or exeditor2 are preferred.
  • nucleotide sequence mutation information for extracting nucleotide sequence mutation information (variant calling) of the full-length genome sequence of the MERS corona virus, one or more methods selected from the group consisting of SOAPsnp, SAMtools, Picard, and GATK may be used, and GATK is preferred.
  • the present invention uses multiple sequence assembly and sequence alignment algorithms, manual and automatic sequencing editing, and sensitive mutation detection and automation methods.
  • the present invention is also a hybrid assembly method used by applying the sequencing method, the assembler, the nucleotide sequence variation information extraction (variant calling) method, and the codon code aligner.
  • the sanger method which was used as a method of sequencing the initial first-generation fragment sequence, separates the DNA fragments and reads them out by radioactivity, which is inconvenient and time-consuming to manipulate.
  • NGS Next Generation Sequencing
  • the NGS decomposes one genome into countless fragments to generate a large number of short-length (100 to 200 nucleotides) reads, read each read at the same time, and then assemble it using computational technology to decode genome information. With this method, it has become possible to perform highly efficient genome sequencing.
  • next-generation sequencing method DNA is extracted from a sample or RNA is extracted, synthesized with cDNA, mechanically fragmented, and then a library having a specific size is produced and used for sequencing.
  • a large-capacity sequencing equipment four kinds of complementary nucleotide binding and separation reactions are repeated with one base unit to produce initial sequencing data, and later, initial data processing (trimming). , Mapping, identification of genomic mutations, and analysis of mutation information, and other analysis steps using bio-informatics.
  • the existing sanger method requires DNA of about 1 kbp template for sequencing, so a library construction and cloning process were required, and a lot of time and cost were consumed for this.
  • the NGS simplifies the process of obtaining a clone, cuts the DNA into short fragments or reads, and then directly amplifies it using a primer, or directly sequence or sequence from a single DNA molecule without amplifying the clone.
  • PCR techniques were applied individually.
  • the read fragment is an assembly type that composes the entire nucleotide sequence based on the nucleotide sequence information, and prior knowledge of the reference relies on the reference assembly used to align the nucleotide sequence information and the reference information.
  • There is a de novo assembly in which the nucleotide sequence information is aligned and recombined to reconstruct the original whole sequence without doing so.
  • the Dinobo assembly is divided into two stages.
  • the steps of creating a contig (assembly sequence) without gaps in the form of adjacent sequences by aligning and overwrapping multiple reads, which are sequence fragments, and connecting one or more of the contigs by ordering and setting the orientation to connect the scaffold It consists of forming (Scaffold).
  • the NGS includes clonal amplification, a massively parallel method that improves efficiency by handling hundreds of thousands of clones at the same time, and a new nucleotide sequence determination method that can be read with a ruler (non-sanger method, base/color calling ) Technology was also introduced.
  • the Sanger Sequencing (Sanger sequencing) amplifies and analyzes the amount of sample through PCR. It is slower than the NGS method, but it is good in terms of accuracy. Was utilized.
  • the human MERS coronavirus used for the present invention was used by pre-sale of isolates in Korea (GenBank registration number KT029139).
  • the MERS coronavirus isolate was inoculated or infected with VERO cells to culture virus cells, and the virus cells were cultured in a carbon dioxide incubator in a biosafety grade 2 extraction culture facility.
  • QIAmp viral RNA minikit Qiagen
  • RNeasy minikit Qiagen
  • human MERS coronavirus RNA MERS-HCoV(KOR) RNA
  • primer candidate groups were selected, and among the primer candidate groups, 53°C ⁇ TM ⁇ 57°C, degeneracy was 2 or less, and Inosine ( Among the primers with 1 or less N base), a total of 14 sets in which an amplicon was generated between 3000 were selected.
  • the “degeneracy” refers to a phenomenon in which one type of amino acid corresponds to a plurality of codons. Due to the degenerate rush, the base sequence of a gene encoding a specific amino acid sequence cannot be determined, but an oligonucleotide including all possible base sequences can be synthesized.
  • a total of 14 selected primer sets of the present invention are shown in Table 1 below.
  • Reverse Transcript reaction was performed using the MERS-HCoV(KOR) RNA with a Random hexamer and Superscript III First-Strand Synthesis SuperMix (Cat No. 18080400). 50ng/ ⁇ l RNA 1 ⁇ l, random hexamer (50ng/ ⁇ l) 1 ⁇ l, annealing buffer 1 ⁇ l, DEPC water 5 ⁇ l into a 0.2 ⁇ l PCR tube, mix lightly, incubate at 65°C for 5 minutes, cool for at least 1 minute on ice, and lightly After centrifugation, it was placed in the same tube, and 2X Reaction mix 10 ⁇ l and 4 ⁇ l Enzyme mix 2 ⁇ l were additionally added and mixed to a total of 20 ⁇ l. After incubation at 25° C. for 10 minutes, reverse transcription was performed at 50° C. for 50 to 90 minutes, and then placed at 85° C. for 5 minutes to inactivate the enzyme.
  • MERS-HCoV(KOR) RNA was subjected to Reverse Transcript (reverse transcription) reaction using Gene Specific Pimer (GSP) and Superscript III First-Strand Synthesis Super Mix. MERS-HCoV in the selected primer set in Table 1 above, set No.
  • the primer set excluding 8 was mixed with 6 even numbered primer sets (set No: 2,4,6,10,12,14) (Mix, odd numbered primer set (Set No: 1,3, 5,7,9,11,13,15,17) After generating Gene Specific Pimer (GSP) by mixing 7 pieces, (optimization) 50ng/ ⁇ l RNA 1 ⁇ l, each of the even numbered primer set or odd numbered primer Gene Specific Pimer (GSP) 1 ⁇ l, annealing buffer 1 ⁇ l, DEPC water 5 ⁇ l generated as a set were put in a 0.2 ⁇ l PCR tube and mixed lightly After incubation at 65°C for 5 minutes, cool on ice for at least 1 minute, lightly centrifugation, and the same Into the tube, 10 ⁇ l of 2X Reaction mix and 2 ⁇ l of 4 ⁇ l Enzyme mix were added and mixed to a total of 20 ⁇ l After incubation at 25°C for 10 minutes, reverse transcription was performed at 50°C for 50 to 90 minutes, and then left at 85°C for 5 minutes.
  • First strand cDNA 2 ⁇ l synthesized by reverse transcription in Example 4 Forward primer 1 ⁇ l, Reverse primer 1 ⁇ l, KAPA hotstart ready mix 10 ⁇ l, 22 ⁇ l, and sterile distilled water were added to 0.2 ⁇ l PCR tube, mixed, and lightly centrifuged and then PCR was performed.
  • the PCR conditions were initial denaturation 98° C. 30 seconds, denaturation 98° C. 30 seconds, annealing 55° C. 30 seconds, extension 72° C. 2 minutes and 30 cycles, followed by final extension 3 minutes.
  • sequencing method was analyzed using Sanger, Sequel (Pacbio, Pacbio RSII), MINion (Oxford Nanopore Technology), and Miseq (Illumina) methods.
  • the MERS-CoV full-length genome (Accession number KT029139) reported in Korea and the sequencing sequence were compared. Sequel (Pacbio RSII), MinION, MiSeq, Sanger sequencing based on the results of the consensus sequence was generated and compared with the reference (reference, Accession number KT029139) strain, consensus and reference were found to be a total of 8 nt inconsistency. This means that there is a variation at the level of microevolution in the experimental strain compared to the reference strain.
  • NGS Next Generation Sequencing analysis data
  • analysis result Sequencing concentration of input RNA(ng/ul) input gDNA(ng) Run time % coverage (variant detection) % accuracy Sanger 50 - - 99.8571 (30065/30108) 99.9700 (30056/30065) MinION 50 1.5ug 8 to 24 hours 99.9136 (30082/30108) 99.9767 (30075/30082) PacBio RSII 50 1.5ug 10 hours 99.8903 (30075/30108) 100 (30075/30075) MiSeq 50 0.5ug 48 hours 99.8106 (30051/30108) 100 (30051/30051)
  • the MERS coronavirus primer detection specificity was confirmed in all selected primer sets except for Set8 as a result of In silico analysis as shown in Table 3 below.
  • the primer set in Table 1 of the present invention has the specificity of the MERS coronavirus, so that the primer set can be used as an oligonucleotide or a probe in a diagnostic method based on hybridization of nucleic acids such as microarrays. have.
  • HCoV-EMC/2012 (a novel MERS coronavirus reported in 2012, reported to infect various animals including bats and pigs. ) was selected as the virus to be tested, and serum of a 16-week-old female mouse mouse and PBS were used. Table 3 below shows the samples used for application of the simulated animal sample.
  • Sample list of simulated animal samples NO Sample Sample One Mouse serum + HCoV-EMC/2012: A serum HCoV-EMC/2012 culture extracted from a 16-week-old female mouse was diluted, and the virus dilution concentration was 4.4 ⁇ 10 5 , 4.4 ⁇ 10 4 , 4.4 ⁇ 10 3 pfu/10 ⁇ l to be.
  • 2 PBS + HCoV-EMC/201 HCoV-EMC/2012 culture was diluted in PBS, and the dilution concentration of the virus was 4.4 ⁇ 10 5 , 4.4 ⁇ 10 4 , 4.4 ⁇ 10 3 pfu/10 ⁇ l.
  • Negative control sterilized tertiary distilled water not inoculated with HCoV-EMC/2012 culture
  • the serum RNA extract is the mouse serum or PBS, and the sample buffer is diluted to each of three concentrations (4.4 ⁇ 10 5 , 4.4 ⁇ 10 4 , 4.4 ⁇ 10 3 pfu/10 ⁇ l).
  • the RNA extract using 10 ⁇ l of the diluted sample was used as a template.
  • As a result of applying PCR using the primer set of Table 1 using the simulated RNA as a template it was confirmed that most of the amplicon (amplified gene) was synthesized at the concentration of 10 5 in both the serum and PBS sample samples. As shown in FIG.
  • a bat MERS (btMERS) corona virus was selected as a similar virus.
  • the virus is Corona Virus Neoromicia, NeoCoV, and the origin of the virus of the present invention is GeneBank Registration Number (Genome Accession number) KC869678.
  • the virus has an isolation source of South Africa Bat, belongs to a species with the MERS coronavirus, and is a sister taxon, has an nt sequence similarity of 85% with the MERS corona virus, and its size is 30108bp.
  • 11 plasmids containing 2940 bp fragments were received, and then plasmids were prepared and cloned as follows.
  • the NeoCov genome fragments were synthesized, ligated, amplified through PCR, and then the nucleotide sequence was confirmed to evaluate versatility.
  • a btMERS (bat-MERS, NeoCoV, bat MERS corona virus) genome or a recombinant vector was constructed.
  • cloning was performed using E. coli as a competent cell, and ampicillin was used as a plasmid selection marker.
  • the plasmid transfection was performed by a heat shock method using high and low temperatures, and the cloning was performed by a cloning method using topoisomerase that does not require ligase.
  • ligation as shown in FIG. 6, plasmids having a size of about 3 kb were concatenated, and each restriction enzyme extension primer was designed and used at both ends for ligation with a size of 6 kb.
  • extension primer contained a restriction enzyme at both ends of the extension, and the same restriction enzyme was made to each fragment to be linked (Fig. 6).
  • extension PCR 2 ⁇ l of the plasmid template, 1 ⁇ l of Forward primer, 1 ⁇ l of Reverse primer, 10 ⁇ l of KAPA hotstart ready mix, and 6 ⁇ l sterile distilled water were added to a 0.2 ⁇ l PCR tube, mixed, and lightly centrifuged, followed by PCR.
  • the PCR conditions were initial denaturation 98°C for 30 seconds, following denaturation 98°C for 30 seconds, annealing 55°C for 30 seconds, extension 72°C for 2 minutes and 30 cycles, and final extension 3 minutes, followed by PCR amplification and purification using Qiagen PCR purification. . Restriction digestion of the end of each generated PCR product was digested using a restriction enzyme for each PCR product.
  • the produced bat MERS corona virus genome fragment (NeoCoV genome fragment, btMERS corona virus genome fragment) was inoculated or infected with VERO cells to cultivate virus cells, and the bat MERS corona virus cells were carbon dioxide incubators in a biosafety class 2 extraction culture facility. Cultured in.
  • QIAmp viral RNA minikit Qiagen
  • RNeasy minikit Qiagen
  • PCR was performed using the selected primer set of the present invention.
  • the 6kb Ligation Fragment 2 ⁇ l, Forward primer 1 ⁇ l, Reverse primer 1 ⁇ l, KAPA hotstart ready mix 10 ⁇ l, and sterile distilled water were added to 0.2 ⁇ l PCR tube, mixed, and lightly centrifuged to perform PCR.
  • the PCR conditions were initial denaturation 98°C for 30 seconds, following denaturation 98°C for 30 seconds, annealing 55°C for 30 seconds, extension 72°C for 2 minutes and 30 cycles, followed by final extension for 3 minutes.
  • Example 6 all sequencing was performed using a PCR product using a selected primer set after RT (reverse transcription) with the random primers of Example 4 above.
  • the Sanger sequencing method used the Primer walking method to sequence all 13 amplicons to obtain a nucleotide sequence. The quality of all the obtained nucleotide sequences were checked by referring to the chromatogram of the bases with Q20 or higher, and after trimming using the ezeditor2 program, they were compared and analyzed with the reference genome using a codoncode aligner.
  • MinION Nanopore sequencing
  • NeoCoV NGS sequencing of NeoCoV was possible only with MinION.
  • the library construction and sequencing protocol used for sequencing the MERS corona virus were performed in the same manner.
  • the purified amplification products of NeoCoV were adjusted to the same concentration, they were mixed so as to have a total amount of 1-1.5 ug and used to prepare the MinION sequencing library.
  • MinION sequencing followed Oxford Nanopore's standard protocol. After MinION progress, a total of 413,513 reads (about 51GB) raw data were calculated, the throughput read length was about 3k, and 51GB of fast5 format raw data calculated by MinION equipment was used as a fastq file using Oxford Nanopore's own program, Guppy software. Converted to.
  • NeoCoV converted to fastq was mapped to a reference genome using a CLC genomic workbench, and assembled, and finally a complete genome sequence consisting of one long contig consensus sequence of about 30 kb was obtained.
  • a complete genome sequence consisting of one long contig consensus sequence of about 30 kb was obtained.
  • the present invention relates to a diagnostic kit and a method for confirming a full-length genome sequence through MERS coronavirus full-length genome amplification, and has industrial applicability.
  • the method, kit, and composition of the present invention makes it possible to obtain the full-length genomic sequence of the MERS coronavirus very accurately and quickly, and through this, it can be effectively applied to the diagnosis of diseases caused by the human MERS coronavirus.

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Abstract

Procédé d'obtention d'une séquence complète du génome pour déterminer la présence d'une infection par un virus MERS-CoV, et utilisation correspondante. Le procédé, le kit et la composition de la présente invention comprennent un ensemble de 14 amorces universelles capables d'amplifier la séquence génomique d'un coronavirus, permettant ainsi une identification facile et rapide de la présence d'un virus cible dans un échantillon et des maladies associées. De plus, 99,9 % de précision et de polyvalence sont confirmés par séquençage du génome entier, et une détection de variants de 99,88 % est confirmée, et ainsi la présence d'un virus MERS-CoV et de maladies associées peut être identifiée de manière commode et rapide. En outre, la présente invention peut être efficacement utilisée dans l'identification de nouveaux virus par détermination, par séquençage du génome entier, de la présence de nouveaux mutants du virus MERS-CoV.
PCT/KR2020/015281 2019-11-04 2020-11-04 Procédé de détection de génome entier utilisant l'amplification du génome entier d'un virus mers-cov, et kit de diagnostic WO2021091211A1 (fr)

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KR10-2019-0139282 2019-11-04

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113430303A (zh) * 2021-06-29 2021-09-24 杭州圣庭医疗科技有限公司 一种基于纳米孔测序仪的23种呼吸道rna病毒快速鉴定方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180052317A (ko) * 2016-11-10 2018-05-18 충북대학교 산학협력단 메르스 코로나바이러스 검출용 프라이머 세트 및 이의 용도

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180052317A (ko) * 2016-11-10 2018-05-18 충북대학교 산학협력단 메르스 코로나바이러스 검출용 프라이머 세트 및 이의 용도

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE NUCLEOTIDE 2 September 2015 (2015-09-02), ANONYMOUS: "Middle East respiratory syndrome coronavirus isolate MERS-CoV/KOR/KNIH/002_05_2015, complete genome", XP055811624, retrieved from NCBI Database accession no. KT029139 *
KIM YOU-JIN, CHO YONG-JOON, KIM DAE-WON, YANG JEONG-SUN, KIM HAK, PARK SUNGHAN, HAN YOUNG WOO, YUN MI-RAN, LEE HAN SAEM, KIM A-REU: "Complete genome sequence of Middle East respiratory syndrome coronavirus KOR/KNIH/002_05_2015, isolated in South Korea", GENOME ANNOUNCEMENTS, vol. 3, no. 4, 27 August 2015 (2015-08-27), XP055811609, DOI: 10.1128/genomeA.00787-15 *
PARK DONGHYUN, HUH HEE JAE, KIM YEON JEONG, SON DAE-SOON, JEON HYO-JEONG, IM EU-HYUN, KIM JONG-WON, LEE NAM YONG, KANG EUN-SUK, KA: "Analysis of intrapatient heterogeneity uncovers the microevolution of Middle East respiratory syndrome coronavirus", MOLECULAR CASE STUDIES, vol. 2, no. 6, 1 November 2016 (2016-11-01), pages a001214, XP055811630, ISSN: 2373-2865, DOI: 10.1101/mcs.a001214 *
YUSOF MOHAMMED FAROUK, QUEEN KRISTA, ELTAHIR YASSIR MOHAMMED, PADEN CLINTON R, AL HAMMADI ZULAIKHA MOHAMED ABDEL HAMEED, TAO YING,: "Diversity of Middle East respiratory syndrome coronaviruses in 109 dromedary camels based on full-genome sequencing, Abu Dhabi, United Arab Emirates", EMERGING MICROBES & INFECTIONS, vol. 6, no. 1, 1 January 2017 (2017-01-01), pages 1 - 10, XP055811628, DOI: 10.1038/emi.2017.89 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113430303A (zh) * 2021-06-29 2021-09-24 杭州圣庭医疗科技有限公司 一种基于纳米孔测序仪的23种呼吸道rna病毒快速鉴定方法

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