WO2021190633A1 - Amorce pour la détection du nouveau coronavirus du sars-cov-2, kit de test, procédé de détection et application correspondante - Google Patents

Amorce pour la détection du nouveau coronavirus du sars-cov-2, kit de test, procédé de détection et application correspondante Download PDF

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WO2021190633A1
WO2021190633A1 PCT/CN2021/083270 CN2021083270W WO2021190633A1 WO 2021190633 A1 WO2021190633 A1 WO 2021190633A1 CN 2021083270 W CN2021083270 W CN 2021083270W WO 2021190633 A1 WO2021190633 A1 WO 2021190633A1
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primer
seq
primer set
cov
sars
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崔占峰
黄巍
王允
王珲
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牛津大学(苏州)科技有限公司
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Publication of WO2021190633A1 publication Critical patent/WO2021190633A1/fr

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    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • 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 disclosure relates to primers for nucleic acid detection and their applications, and in particular to primers, primer sets, kits, detection methods, and applications for SARS-CoV-2 detection.
  • the currently circulating new coronavirus SARS-CoV-2 (also known as 2019-nCoV, HCoV-19 or new coronavirus) has caused a large number of people to be infected with the new coronavirus pneumonia COVID-19 (also known as "new coronary pneumonia").
  • COVID-19 also known as "new coronary pneumonia”
  • the incubation period of COVID-1 new coronary pneumonia is generally 1 to 14 days, mostly 3 to 7 days.
  • the symptoms are mainly fever, dry cough, and fatigue. Severely ill patients often have breathing difficulties and/or one week after the onset of illness. Hypoxemia, in severe cases, can rapidly progress to acute respiratory distress syndrome, septic shock, difficult to correct metabolic acidosis, coagulation dysfunction, and multiple organ failure.
  • WHO In order to curb the spread of the virus, WHO urgently requires the expansion of screening and testing of potential patients.
  • Viral nucleic acid detection that is, the detection of viral RNA
  • the most common nucleic acid diagnosis method is based on real-time RT-PCR to detect the positive nucleic acid of the new coronavirus.
  • China's BGI https://www.bgi.com/kit
  • the US Centers for Disease Control and Prevention CDC, https://www.cdc.gov/coronavirus/2019-ncov/about/testing. html
  • the methods for detecting SARS-CoV-2 are either based on the use of reverse transcription real-time polymerase chain reaction (RT-qPCR) technology to detect the viral RNA itself, or based on the specific immunoglobulins IgM and IgM that appear in the patient’s blood a few days after infection.
  • RT-qPCR reverse transcription real-time polymerase chain reaction
  • these existing methods require laboratories to be equipped with specialized testing equipment, operated by skilled scientists and technicians, and take a long time (about 2 hours or more), therefore, limit nucleic acid testing in the current global COVID -Widely used in the huge testing needs of the 19 pandemic.
  • the existing nucleic acid detection reagents also have the problem of a high false negative rate (the positive detection rate can be as low as 30% to 50%).
  • the present disclosure provides a rapid, simple, and highly specific SARS-CoV-2 virus detection method.
  • the first aspect of the present disclosure provides an oligonucleotide primer set (hereinafter referred to as the N15 primer set) for amplifying the SARS-CoV-2 gene, particularly preferably its nucleocapsid protein gene region, including : Forward primer: SEQ ID No. 1; Reverse primer: SEQ ID No. 2; Forward inner primer: SEQ ID No. 3; Reverse inner primer: SEQ ID No. 4; Forward loop primer: SEQ ID No.5; and reverse loop primer: SEQ ID No.6.
  • the N15 primer set for amplifying the SARS-CoV-2 gene, particularly preferably its nucleocapsid protein gene region, including : Forward primer: SEQ ID No. 1; Reverse primer: SEQ ID No. 2; Forward inner primer: SEQ ID No. 3; Reverse inner primer: SEQ ID No. 4; Forward loop primer: SEQ ID No.5; and reverse loop primer: SEQ ID No.6.
  • an oligonucleotide primer set for amplifying the SARS-CoV-2 gene, particularly preferably its Orf1ab gene region, including: forward Primer: SEQ ID No. 7; Reverse primer: SEQ ID No. 8; Forward inner primer: SEQ ID No. 9; Reverse inner primer: SEQ ID No. 10; Forward loop primer: SEQ ID No. 11 ; And reverse loop primer: SEQ ID No. 12.
  • an oligonucleotide primer set for amplifying the SARS-CoV-2 gene, particularly preferably its nucleocapsid protein gene region, including : Forward primer: SEQ ID No. 13; Reverse primer: SEQ ID No. 14; Forward inner primer: SEQ ID No. 15; Reverse inner primer: SEQ ID No. 16; Forward loop primer: SEQ ID No. 17; and reverse loop primer: SEQ ID No. 18.
  • an oligonucleotide primer set for amplifying the SARS-CoV-2 gene, particularly preferably its spike protein gene region, including: positive Forward primer: SEQ ID No. 19; Reverse primer: SEQ ID No. 20; Forward inner primer: SEQ ID No. 21; Reverse inner primer: SEQ ID No. 22; Forward loop primer: SEQ ID No. 23; and reverse loop primer: SEQ ID No. 24.
  • a primer set set for amplifying the SARS-CoV-2 gene is also provided, which includes a combination of any two or more of the aforementioned oligonucleotide primer sets.
  • kits for detecting SARS-CoV-2 includes any of the aforementioned oligonucleotide primer sets or primer sets.
  • a method for detecting SARS-CoV-2 using any one of the above-mentioned oligonucleotide primer sets or primer sets includes: obtaining a sample to be tested; extracting a sample to be tested RNA as a template; reverse transcription of the RNA and use any of the above-mentioned oligonucleotide primer sets or primer set sets to amplify the DNA obtained by reverse transcription, or use any of the above-mentioned oligonucleotide primers A group or a set of primer sets performs reverse transcription amplification on the RNA; and based on the amplification result or the reverse transcription amplification result, it is determined whether the sample to be tested contains SARS-CoV-2.
  • oligonucleotide primer sets or primer sets in preparing reagents for detecting SARS-CoV-2 is also provided.
  • the present disclosure also provides a method for detecting nucleic acid in one step, the method comprising: obtaining a sample to be tested, and collecting cells from the sample to be tested; using primers suitable for detecting the nucleic acid, The cells are performed in one step in the same reaction system: cell lysis; nucleic acid extraction; gene amplification or reverse transcription amplification; and based on the amplification result, it is determined whether the sample to be tested contains the nucleic acid.
  • the present disclosure also provides the use of the aforementioned oligonucleotide primer set, or primer set collection, or kit, or detection method for diagnosing SARS-CoV-2 related diseases or symptoms.
  • Figure 1 shows the RNA profile of the SARS-CoV-2 virus and related gene regions designed according to the N15, O117, N1, and S17 primer sets described in the first to fourth aspects of the present disclosure
  • Figure 2 shows the gel electrophoresis and fluorescence results of the in vitro LAMP reaction of the N15, O117, N1, and S17 primer sets on SARS-CoV-2 synthesized DNA fragments in an embodiment according to the present disclosure, in which:
  • Figure 2A shows the gel electrophoresis and fluorescence results of N1 primer set (target is N gene in vitro transcribed DNA): (1) N1 primer set + N gene DNA (200k copies); (2) N1 primer set + N gene DNA (200 Copies); (3) N1 primer set + N gene DNA (200k copies) + human genome; (4) N1 primer set + N gene DNA (200 copies) + human genome; (5) N1 primer set + human genome; ( 6) Human ⁇ -actin primer + human genome; (7) Human ⁇ -actin primer + N gene DNA (200k copies); and (L) pre-stained protein Ladder;
  • Figure 2B shows the gel electrophoresis and fluorescence results of N15 primer set (target is N gene in vitro transcribed DNA): (1) N15 primer set + N gene DNA (200k copies); (2) N15 primer set + N gene DNA (200 Copies); (3) N15 primer set + N gene DNA (200k copies) + human genome; (4) N15 primer set + N gene DNA (200 copies) + human genome; (5) N15 primer set + human genome; ( 6) Human ⁇ -actin primer + human genome; (7) Human ⁇ -actin primer + N gene DNA (200k copies); and (L) pre-stained protein Ladder;
  • Figure 2C shows the gel electrophoresis results of O117 primer set (target is Orf1ab gene in vitro transcription DNA): (1) O117 primer set + Orf1ab gene DNA (200k copies); (2) O117 primer set + Orf1ab gene DNA (200 copies) ; (3) O117 primer set + Orf1ab gene DNA (20 copies); (4) O117 primer set + Orf1ab gene DNA (2 copies); (5) O117 primer set + Orf1ab gene DNA (200 copies) + human genome; ( 6) O117 primer set + human genome; (7) O117 primer set + water (8) human ⁇ -actin primer + human genomic DNA; and (L) pre-stained protein Ladder;
  • Figure 2D shows the gel electrophoresis results of S17 primer set (target is S gene in vitro transcription DNA): (1) S17 primer set + S gene DNA (200k copies); (2) S17 primer set + S gene DNA (200 copies) ; (3) S17 primer set + S gene DNA (20 copies); (4) S17 primer set + S gene DNA (2 copies); (5) S17 primer set + S gene DNA (200 copies) + human genome ; (6) S17 primer set + human genome; (7) S17 primer set + water (8) human ⁇ -actin primer + human genomic DNA; and (L) pre-stained protein Ladder;
  • Figure 3 shows the N1 and N15 primer sets in an in vitro RT-LAMP reaction with SARS-CoV-2 virus N gene RNA as the target in an embodiment according to the present disclosure, respectively, in ( Figure 3A) 15 minutes, ( Figure 3B) Gel electrophoresis results of the reaction mixture sampled at 20 minutes and ( Figure 3C) 30 minutes: (1) N1 primer set + N gene RNA (200 copies); (2) N1 primer set + N gene RNA (20 Copies); (3) N1 primer set + N gene RNA (2 copies); (4) N15 primer set + N gene RNA (200 copies); (5) N15 primer set + N gene RNA (20 copies); (6) ) N15 primer set + N gene RNA (2 copies); (L) pre-stained protein Ladder;
  • Figure 4 shows an example according to the present disclosure
  • the O117 primer set uses the SARS-CoV-2 virus Orf1ab gene RNA as the target ( Figures 4A, 4B, 4C)
  • the S17 primer set uses the SARS-CoV-2 virus S In the in vitro RT-LAMP reaction with gene RNA as the target ( Figure 4D, 4E, 4F)
  • samples were taken at ( Figure 4A, 4D) 15 minutes, ( Figure 4B, 4E) 20 minutes, and (Figure 4C, 4F) 30 minutes.
  • Figure 5 shows the color observation and gel electrophoresis results of the colorimetric RT-LAMP reaction using the N15 primer set in an embodiment according to the present disclosure.
  • Figure 5A shows the color of the reaction mixture before RT-LAMP.
  • 5B is the color of the reaction mixture after RT-LAMP
  • Figure 5C is the gel electrophoresis of the reaction mixture, and (1) N15 primer set + N gene RNA (200k copies); (2) N15 primer set + N gene RNA (200 Copies); (3) N15 primer set + N gene RNA (20 copies); (4) N15 primer set + N gene RNA (2 copies); (5) N15 primer set + human genome; (6) human primer + human Genome; (7) human primer + whole human RNA; and (8) only colorimetric dye MasterMix;
  • Fig. 6 shows the results of an in vitro experiment on the RT-LAMP colorimetric/fluorescence detection of SARS-CoV-2 using the fluorescently labeled primer set described in the present disclosure in an embodiment according to the present disclosure, where Fig. 6A is The colorimetric results of the amplified products show that Figure 6B is the fluorescence image of the product under UV light, and test tubes #1, #2, and #3 contain FAM fluorescently labeled N15 primer set, O117 primer set and human ⁇ muscle, respectively. Kinesin primer set (SEQ ID No. 29-32).
  • Figure 7 shows the RT-LAMP reaction test results of the one-step method for detecting the virus to be tested in an embodiment of the present disclosure using human cell lines and human primers in an embodiment according to the present disclosure
  • the cells are dispersed in Hank's Balanced Salt Solution (HBSS), and each contains (1) 0 cells + human ⁇ -actin primers; (2) 10 cells + human ⁇ -actin primers; (3) 50 cells + human ⁇ -actin primer; (4) 100 cells + human ⁇ -actin primer; (5) hRNA + human ⁇ -actin primer; (6) water + human ⁇ - Actin primers; (7) water (no primers); and
  • cells are dispersed in 0.85% NaCl solution, and each contains (1) 0 cells + human ⁇ -actin primers; (2) 10 Cell + human ⁇ -actin primer; (3) 50 cells + human ⁇ -actin primer; (4) 100 cells + human ⁇ -actin primer; (5) hRNA + human ⁇ -actin Primers; and (6) water + human
  • the present disclosure provides a rapid, simple, and highly specific SARS-CoV-2 virus detection method, which can be used to quickly and accurately determine patients with new coronary pneumonia (including patients with new coronary pneumonia who have not yet had an immune response) and asymptomatic carriers And determine whether the SARS-CoV-2 virus exists in other situations (such as environmental samples from living places).
  • a plurality of oligonucleotide primer sets are provided for amplifying the SARS-CoV-2 gene.
  • the first aspect provides an N15 primer set for amplifying the SARS-CoV-2 gene, particularly preferably its nucleocapsid protein gene region.
  • the primer set includes: forward primer: SEQ ID No. 1; reverse primer : SEQ ID No. 2; Forward inner primer: SEQ ID No. 3; Reverse inner primer: SEQ ID No. 4; Forward loop primer: SEQ ID No. 5; and Reverse loop primer: SEQ ID No. 6.
  • an O117 primer set for amplifying the SARS-CoV-2 gene, particularly preferably its Orf1ab gene region.
  • the primer set includes: forward primer: SEQ ID No. 7; reverse primer: SEQ ID No. 8; forward inner primer: SEQ ID No. 9; reverse inner primer: SEQ ID No. 10; forward loop primer: SEQ ID No. 11; and reverse loop primer: SEQ ID No. 12.
  • the third aspect provides an N1 primer set for amplifying the SARS-CoV-2 gene, particularly preferably its nucleocapsid protein gene region.
  • the primer set includes: forward primer: SEQ ID No. 13; reverse primer : SEQ ID No. 14; Forward inner primer: SEQ ID No. 15; Reverse inner primer: SEQ ID No. 16; Forward loop primer: SEQ ID No. 17; and Reverse loop primer: SEQ ID No. 18.
  • an S17 primer set for amplifying the SARS-CoV-2 gene, particularly preferably its spike protein gene region.
  • the primer set includes: forward primer: SEQ ID No. 19; reverse primer: SEQ ID No. 20; Forward inner primer: SEQ ID No. 21; Reverse inner primer: SEQ ID No. 22; Forward loop primer: SEQ ID No. 23; and Reverse loop primer: SEQ ID No. 24 .
  • the 5'end of the forward inner primer sequence of the oligonucleotide primer set of any of the above aspects may have a fluorescent label.
  • the fluorescent label may be FAM.
  • a primer set set for amplifying the SARS-CoV-2 gene is also provided, which includes a combination of any two or more of the aforementioned oligonucleotide primer sets.
  • the method of using any of the above-mentioned oligonucleotide primer sets or primer sets for amplification can be any gene amplification technology known to those skilled in the art, such as, but not limited to, polymerase Chain reaction (Polymerase Chain Reaction, PCR), multiplex polymerase chain reaction (Multiplex PCR, mPCR), real-time or quantitative polymerase chain reaction (Real-Time/Quantitative PCR, qPCR), nucleic acid sequence-dependent amplification ( Nucleic Acid Sequence-Based Amplification (NASBA) and Loop-Mediated Isothermal Amplification (LAMP), Reverse-Transcription Polymerase Chain Reaction (RT-PCR), reverse Record multiple polymerase chain reaction (RT-mPCR), reverse transcription real-time or quantitative polymerase chain reaction (RT-qPCR), reverse transcription nucleic acid sequence dependent amplification (RT-NASBA) and reverse transcription loop-mediated Guided isothermal amplification (RT-LAMP), as long as it can achieve the
  • the method of amplification using the oligonucleotide primer set or primer set set of any of the above aspects may be loop-mediated isothermal amplification (LAMP) or reverse transcription loop-mediated isothermal amplification (RT-LAMP).
  • LAMP loop-mediated isothermal amplification
  • RT-LAMP reverse transcription loop-mediated isothermal amplification
  • kits for detecting SARS-CoV-2 includes the oligonucleotide primer set or primer set set of any one of the above aspects.
  • the primer set included in the kit may be the primer set of the first aspect and the second aspect of the disclosure.
  • the kit may also include a DNA polymerase, and optionally a pH indicator.
  • the kit may also include DNA polymerase and reverse transcriptase, and optionally a pH indicator.
  • the kit may also include a single enzyme with dual functions of RNA reverse transcriptase and DNA polymerase.
  • the kit may also include a gene amplification reaction solution or premix, a negative control, quality control materials, and/or instructions for use.
  • the gene amplification reaction solution may include water, dNTPs, DNA polymerase (such as Taq enzyme), buffer, and MgCl 2 .
  • the gene amplification reaction solution or premix solution may also include reverse transcriptase.
  • the gene amplification reaction solution or premix solution may also include an enzyme stabilizer, a fluorescent dye (such as an electrophoresis fluorescent dye), a pH indicator, and the like.
  • the negative control may be a human actin primer. In some of these embodiments, the negative control is a human ⁇ -actin primer. In some embodiments, the human ⁇ -actin primer may include: forward primer: SEQ ID No. 29; reverse primer: SEQ ID No. 30; forward internal primer: SEQ ID No. 31; Reverse inner primer: SEQ ID No. 32.
  • a method for detecting SARS-CoV-2 using any one of the above-mentioned oligonucleotide primer sets or primer sets may include: obtaining a sample to be tested; extracting a sample to be tested RNA as a template; reverse transcription of the RNA and use any of the above-mentioned oligonucleotide primer sets or primer sets to amplify the DNA obtained by reverse transcription, or use any of the above-mentioned oligonucleotides A primer set or a set of primer sets performs reverse transcription amplification on the RNA; and based on the amplification result or reverse transcription amplification result, it is determined whether the sample to be tested contains SARS-CoV-2.
  • oligonucleotide primer sets or primer sets in preparing reagents for detecting SARS-CoV-2 is also provided.
  • the detection of SARS-CoV-2 may include: obtaining a sample to be tested; extracting RNA of the sample to be tested as a template; performing reverse transcription on the RNA and using any of the above-mentioned oligonucleotides A primer set or primer set set is used to amplify the DNA obtained by reverse transcription, or any one of the above-mentioned oligonucleotide primer sets or primer set sets are used to perform reverse transcription amplification of the RNA; and based on the amplification As a result or reverse transcription amplification result, it is determined whether the sample to be tested contains SARS-CoV-2.
  • the present disclosure also provides a method for detecting nucleic acid in one step, the method comprising: obtaining a sample to be tested, and collecting cells from the sample to be tested; using primers suitable for detecting the nucleic acid, The cells are performed in one step in the same reaction system: cell lysis; nucleic acid extraction; gene amplification or reverse transcription amplification; and based on the amplification result, it is determined whether the sample to be tested contains the nucleic acid.
  • the nucleic acid may be DNA. In some of these embodiments, the nucleic acid may be RNA. In some of these embodiments, the amplification may be LAMP amplification. In some of these embodiments, the reverse transcription amplification may be RT-LAMP amplification.
  • the nucleic acid may be viral RNA
  • the method may include: obtaining a sample to be tested, and collecting cells from the sample to be tested; achieving cell lysis and RNA extraction in one step in the same reaction system , RNA reverse transcription and DNA amplification; and, based on the amplification result, determine whether the test sample contains the test virus.
  • the sample to be tested may be a clinical virus specimen, such as a throat swab or a nasal swab.
  • the amplification in the seventh, eighth, and ninth aspects may be LAMP, and the reverse transcription amplification may be RT-LAMP.
  • the method of determining whether the sample to be tested contains SARS-CoV-2 based on the amplification result or the reverse transcription amplification result in the seventh, eighth, and ninth aspects described above may be It is through gel electrophoresis, fluorescent dyes or pH indicators, or other qualitative, quantitative, or semi-quantitative methods for the amplification results.
  • the amplification result is confirmed by a pH indicator.
  • the present disclosure also provides the use of the aforementioned oligonucleotide primer set, or primer set collection, or kit, or detection method for diagnosing SARS-CoV-2 related diseases or symptoms.
  • the inventors of the present disclosure have creatively designed primers for multiple specific gene regions of the SAV-CoV-2 virus, and developed a high-specificity primer set that can quickly, simply and sensitively detect SAV-CoV-2.
  • the primer has extremely high detection accuracy and sensitivity.
  • the inventor also overcomes the complex and difficult technical problems of LAMP primer design (Law, JWF, Ab Mutalib, NS, Chan, KG, and Lee, LH (2015) Rapid methods for the detection of foodborne bacterial pathogens: principles, applications , advantages and limits, Frontiers in Microbiology 5.), and added loop primers for acceleration of LAMP reaction, thus making this highly specific primer set applicable to LAMP and RT-LAMP reactions, by performing high-speed amplification of viral RNA Increase, greatly shorten the detection time of SAV-CoV-2, and significantly reduce the detection limit, thus significantly improving the efficiency and sensitivity of SAV-CoV-2 detection.
  • LAMP primer design Law, JWF, Ab Mutalib, NS, Chan, KG, and Lee, LH (2015) Rapid methods for the detection of foodborne bacterial pathogens: principles, applications , advantages and limits, Frontiers in Microbiology 5.
  • the result can also be judged with the naked eye through color development (for example, pH indicator), so that it can be simple and fast without special laboratory equipment or experiment personnel. Determine the test results on the spot.
  • color development for example, pH indicator
  • the inventors of the present disclosure also provide a COVID-19 detection kit using the primer set of the present disclosure, which can quickly, simply and sensitively detect SAV-CoV-2 without requiring complicated instruments, and optionally with naked eyes Read the test results, which can be applied to scenarios such as airports, train stations and hospitals, especially regional hospitals and medical centers in rural areas, to quickly determine patients with new coronary pneumonia (including patients with new coronary pneumonia who have not even developed an immune response) and asymptomatic Carriers, or to determine whether the SAV-CoV-2 virus exists in homes or public places, etc., provide vital information for timely treatment of patients, public health decision-making, and containment of the spread of the virus.
  • Regional hospitals and medical centers in rural areas have paved the way for large-scale SARS-CoV-2 virus screening.
  • the inventors of the present disclosure designed a highly specific primer sequence for the SARS-CoV-2 viral genome sequence published in NCBI (GenBank, NC_045512.2).
  • the present disclosure has designed 4 primer sets, namely O117, S17, N1 and N15 primer sets, which are specific to different regions in the viral gene sequence.
  • Each primer set contains 6 primers, namely forward primer F3, reverse primer B3, forward inner primer FIP, reverse inner primer BIP, forward loop primer LF and reverse loop primer LB.
  • the primer design uses the primer design software PrimerExplorer ( http://primerexplorer.jp/e/ ) as an auxiliary tool.
  • the primer design of the present disclosure considers the key characteristics highly related to the SARS-CoV-2 viral RNA itself to ensure the high specificity, accuracy and sensitivity of each primer set.
  • SARS-CoV-2 is a single-stranded RNA virus of approximately 30 kb.
  • the primer set of the present disclosure specifically targets the genes encoding Open Reading Frame 1ab (Open Reading Frame 1ab, or Orf1ab), Spike Glycoprotein (or S protein), and nuclear genes in SARS-CoV-2 viral RNA.
  • the Orf1ab of SARS-CoV-2 is approximately 21 kb and encodes a replicase polyprotein.
  • the inventors designed an O117 primer set for Orf1ab to cover the reverse region at the 5'end of the viral RNA ( Figure 1).
  • the S17 primer set targets the S gene.
  • the spike glycoprotein encoded by this gene is a key factor for the SARS-CoV-2 virus to bind to human angiotensin converting enzyme 2 (Angiotensin I Converting Enzyme 2, ACE2 protein) and invade human cells.
  • the N gene of the nucleocapsid protein is located at the 3'end of the viral RNA, which is a conserved sequence of SARS-like coronavirus; during the sampling and RNA extraction process, the viral RNA will be attacked by RNase and degrade from the 5'end to the 3'end.
  • the inventors also designed N1 and N15 primer sets for this region, and the primer sets can detect the partially degraded RNA of SARS-CoV-2.
  • the SARS-CoV-2 virus RNA region targeted by each primer set of the present disclosure is 240-260 bp, which can be detected by a gene amplification reaction.
  • the primer design of the present disclosure is also suitable for the application of LAMP and RT-LAMP reactions.
  • the LAMP and RT-LAMP reactions are carried out at a constant temperature (usually 65°C).
  • the DNA amplification is completed in just 30 minutes, and the required equipment is very simple.
  • SARS-CoV-2 is an RNA virus of approximately 30 kb
  • the common single reaction of RT and LAMP can significantly shorten the reaction time and omit RNA purification steps, so that SARS-CoV-2 can be quickly detected.
  • the sequence of the primer set designed by the present disclosure is shown in Table 1 below.
  • the 5'end of the forward inner primer FIP of each primer set can be fluorescently labeled (such as FAM labeled) to quickly and conveniently observe the detection result.
  • the 5'end of the forward inner primer FIP is fluorescently labeled with FAM.
  • Example 2 In vitro detection simulation experiment of SARS-CoV-2 virus synthetic DNA fragment target
  • This embodiment is used to test the primers of the present disclosure for in vitro (in vitro) detection simulation experiments for SARS-CoV-2 synthetic DNA fragments.
  • N, S, and Orf1ab target genes were designed. Each fragment contains a T7 promoter for in vitro transcription, namely N1-T7, N15-T7, S17-T7 , O117-T7, its sequence (SEQ ID NO.25-28) is shown in Table 2.
  • the human ⁇ -actin primer (Poon LLM, et al. Detection of human influenza A viruses by loop-mediated isothermal amplification. Journal of Clinical Microbiology 43,427-430 (2005)) was used as a negative control, and its sequence (SEQ ID NO .29-32) As shown in Table 3. All designed primers and DNA fragments, and the positive control plasmid (2019-nCoV_N_Positive Control) were prepared by Integrated DNA Technologies (IDT, UK).
  • N, S and Orf1ab target genes were prepared synthetically by IDT, and reproduced to 50ng/ ⁇ l according to the manufacturer's instructions.
  • the copy number of each target gene was determined based on its molecular weight, and diluted to 200,000 copies/ ⁇ l, 200 copies/ ⁇ l, 0 copy/ ⁇ l and 2 copies/ ⁇ l for subsequent experiments.
  • T7_N, T7_S and T7_O were prepared synthetically by IDT and reproduced to 50ng/ ⁇ l according to the manufacturer's instructions.
  • the experimental treatment groups of O117, S17, N1 and N15 primer sets are respectively set as follows, where 200k (200,000), 200, 20, and 2 represent the total number of copies of the DNA sequence in the reaction mixture:
  • N1 primer set target is N gene synthetic DNA fragment
  • N1 primer set + N gene DNA 200k copies
  • N1 primer set + N gene DNA 200 copies
  • N1 primer Set + N gene DNA 200k copies
  • N1 primer set + N gene DNA 200 copies
  • N1 primer set + N gene DNA 200 copies
  • N1 primer set + human genome (5)
  • N1 primer set + human genome (6)
  • Human ⁇ -actin Primer + human genome (7)
  • human ⁇ -actin primer + N gene DNA (200k copies) and
  • N15 primer set (target is N gene synthetic DNA fragment): (1) N15 primer set + N gene DNA (200k copies); (2) N15 primer set + N gene DNA (200 copies); (3) N15 primer Set + N gene DNA (200k copies) + human genome; (4) N15 primer set + N gene DNA (200 copies) + human genome; (5) N15 primer set + human genome; (6) Human ⁇ -actin Primer + human genome; (7) human ⁇ -actin primer + N gene DNA (200k copies); and (L) pre-stained protein Ladder.
  • C.O117 primer set (target is Orf1ab gene synthetic DNA fragment): (1) O117 primer set + Orf1ab gene DNA (200k copies); (2) O117 primer set + Orf1ab gene DNA (200 copies); (3) O117 primer Set + Orf1ab gene DNA (20 copies); (4) O117 primer set + Orf1ab gene DNA (2 copies); (5) O117 primer set + Orf1ab gene DNA (200 copies) + human genome; (6) O117 primer set + Human genome; (7) O117 primer set + water; (8) human ⁇ -actin primer + human genomic DNA; and (L) pre-stained protein Ladder.
  • S17 primer set target is S gene synthetic DNA fragment
  • S17 primer set + S gene DNA 200k copies
  • S17 primer set + S gene DNA 200 copies
  • S17 primer Set + S gene DNA (20 copies)
  • S17 primer set + S gene DNA (2 copies)
  • S17 primer set + S gene DNA 2 copies
  • S17 primer set + S gene DNA 2 copies
  • S17 primer set + S gene DNA 2 copies
  • S17 primer set + S gene DNA 2 copies
  • S17 primer set + S gene DNA 2 copies
  • S17 primer set + S gene DNA 200 copies
  • S17 primer Group + human genome (7)
  • S17 primer set + water (8) human ⁇ -actin primer + human genomic DNA; and (L) pre-stained protein Ladder.
  • a 10X primer mixture (FIP, 16 ⁇ M; BIP, 16 ⁇ M; F3, 2 ⁇ M; B3, 2 ⁇ M; LF, 4 ⁇ M; LB, 4 ⁇ M) was prepared using the O117, S17, N1, and N15 primer sets of the present disclosure, respectively.
  • fluorescent dyes New England Biolabs, UK were added to the N1 and N15 treatment groups, and the color changes were directly observed with the naked eye from the PCR tube under ultraviolet irradiation, and compared with the gel electrophoresis results.
  • the N1 and N15 primer sets amplified the DNA of the N gene (lanes 1-2 in Figure 2A and Figure 2B), but did not amplify the human genomic DNA ( Lane 5 in Figure 2A and Figure 2B).
  • O117 and S17 primer sets also specifically amplify the DNA of the Orf1ab gene and S gene, respectively (lanes 1-4 in Figure 2C and Figure 2D), but did not amplify the human genome DNA (lane 5 in Figures 2C and 2D). It can be seen that the O117, S17, N1 and N15 primer sets have superior amplification specificity for the SARS-CoV-2 synthetic DNA fragment target compared to the human genome.
  • each primer set to the corresponding gene in the reaction mixture (lane 4 in Figures 2A and 2B, lane 5 in Figure 2C and 2D) is not affected by interference. It shows the excellent anti-interference ability of the primer set of the present disclosure, which is an indispensable property for detecting viral RNA from human samples using LAMP.
  • the PCR tubes were observed under ultraviolet light. It can be seen that although there is a fluorescent background in the amplification negative tubes ( Figure 2A and 2B), the amplification positive tubes can still be seen The fluorescence intensity is stronger than the fluorescence intensity in the amplified negative tube. The fluorescence report result is consistent with the result of the electrophoresis gel.
  • all LAMP reactions for DNA amplification are 30 minutes. It can be seen that each primer set can fully amplify at least 200 copies of the corresponding gene sequence within 30 minutes and observe obvious positive results.
  • the O117 and S17 primer sets can even fully amplify viral DNA containing only as few as 20 and 2 copies within 30 minutes (lane 3 in Figure 2C, lane 4 in Figure 2D), and show positive results, which indicates The primer set of the present disclosure has high detection efficiency and detection sensitivity.
  • Example 3 In vitro simulation experiment of SARS-CoV-2 viral RNA target
  • This embodiment is an in vitro simulation experiment used to test the primers of the present disclosure for detecting SARS-CoV-2 viral RNA.
  • T7 High Yield RNA synthesis HiScribe kit (HiScribe TM T7High Yield RNA Synthesis Kit , New England Biolabs, UK), in the Example 1 of the embodiment of the DNA sequence in vitro transcription synthesis IDT T7_N, T7_S and the T7_O.
  • the RNeasy Mini Kit kit (Qiagen, UK) was used to purify the transcription product, and the NanoDrop nucleic acid detector was used to measure the concentration and quality of RNA.
  • the copy number of each target RNA was determined based on its molecular weight, and diluted to 200,000 copies/ ⁇ l, 200 copies/ ⁇ l, 20 copies/ ⁇ l and 2 copies/ ⁇ l for subsequent experiments.
  • the RNeasy Mini Kit kit (Qiagen, UK) was used to purify human whole RNA from human iPSC cells.
  • the experimental treatment groups of O117, S17, N1 and N15 primer sets are respectively set as follows, where 200k (200,000), 200, 20, and 2 represent the total number of copies of the DNA sequence in the reaction mixture:
  • N1 and N15 primer sets in vitro transcribed RNA targeting N gene: (1) N1 primer set + N gene RNA (200 copies); (2) N1 primer set + N gene RNA (20 copies); (3) ) N1 primer set + N gene RNA (2 copies); (4) N15 primer set + N gene RNA (200 copies); (5) N15 primer set + N gene RNA (20 copies); (6) N15 primer set + N gene RNA (2 copies); (L) pre-stained protein Ladder.
  • O117 and S17 primer set in vitro transcribed RNA targeting N gene: (1) O117 or S17 primer set + RNA target (200k copies); (2) O117 or S17 primer set + RNA target (200 copies); (3) O117 or S17 primer set + RNA target (20 copies); (4) O117 or S17 primer set + RNA target (2 copies); (5) O117 or S17 primer set + RNA target (200 copies) + human genome ; (6) O117 or S17 primer set + human genome; (7) O117 or S17 primer set + water; (8) human primer + human genome.
  • the experimental procedure of this example is basically the same as that of example 2, but at 15 minutes, 20 minutes, and 30 minutes, the reaction mixture is sampled and gel electrophoresis is performed to confirm the amplification result.
  • the sensitivity of RT-LAMP was evaluated by further diluting the number of copies of the RNA target in each reaction from 200 to 2 (the total volume of the reaction solution is 25 ⁇ l), and by 15 minutes, 20 minutes after the reaction started. The reaction mixture was sampled at 30 minutes and 30 minutes to check the reaction efficiency and detection limit.
  • Experimental results show that after integrating RT and LAMP into one reaction, O117, S17, N1 and N15 primer sets can still amplify viral RNA fragments with high specificity and sensitivity.
  • both N1 and N15 primer sets can detect 2 copies of target RNA within 20 minutes and 30 minutes ( Figures 3A, 3B and 3C).
  • the S17 primer set can detect 2 copies of S gene RNA in 30 minutes of RT-LAMP reaction
  • the O117 primer set can detect 20 copies of Ord1ab gene RNA in 30 minutes.
  • the results show that the O117, S17, N1, and N15 primer sets of the present disclosure are all very sensitive, and viral RNA can be detected in a 30-minute RT-LAMP reaction.
  • the use of primers N1 and S17 can achieve a sensitivity of 80 copies of viral RNA per milliliter.
  • Example 4 In vitro experiment of RT-LAMP colorimetric detection of SARS-CoV-2
  • This embodiment is used to test the in vitro experiment of RT-LAMP colorimetric detection of SARS-CoV-2 viral RNA using the primer set of the present disclosure (take N15 as an example).
  • the experimental treatment groups used in this example are as follows, where 200k (200,000), 200, 20, and 2 represent the total number of copies of the DNA sequence in the reaction mixture: (1) N15 primer set + N gene RNA (200k copies); 2) N15 primer set + N gene RNA (200 copies); (3) N15 primer set + N gene RNA (20 copies); (4) N15 primer set + N gene RNA (2 copies); (5) N15 primer set + Human genome; (6) human primer + human genome; (7) human primer + fully human RNA; and (8) only the colorimetric dye MasterMix.
  • RNA target acquisition and purification methods and RT-LAMP experimental steps used in this example are all as described in Example 3, but the WarmStart TM LAMP 2X Master Mix (DNA&RNA) master mix is replaced with or WarmStart TM Colorimetric LAMP 2X Master Mix (DNA&RNA) master mix.
  • DNA&RNA WarmStart TM LAMP 2X Master Mix
  • DNA&RNA WarmStart TM Colorimetric LAMP 2X Master Mix
  • a pH indicator can be used to show positive or negative results of RT-LAMP.
  • the pH indicator may be phenol red, which is pink at pH 8.2-8.6, and turns yellow (positive) when the pH drops.
  • Figure 5A shows the color of the reaction mixture before and after the 30-minute RT-LAMP reaction, and compares it with the gel electrophoresis result (Figure 5B).
  • Figure 5A if the target sequence has been amplified (amplification positive), a visible color change from pink to yellow can be observed.
  • the intensity of the RT-LAMP product in the gel as shown in Figure 5B is consistent with the color change of the reaction mixture in Figure 5A. Therefore, the colorimetric RT-LAMP assay can reliably detect the N gene of SARS-CoV-2 viral RNA, and the color change can semi-quantitatively indicate the number of target sequences. As a result, RT-LAMP results can be visualized, so that the results can be read with the naked eye without the need for special equipment and fluorescent dyes.
  • Example 5 In vitro experiment of RT-LAMP colorimetric/fluorescence detection of SARS-CoV-2 with fluorescently labeled primers
  • This example is used to test the use of the primer set of the present disclosure (taking N15 and O117 as examples) to perform FAM fluorescent labeling (5'-FAM-FIP) on the 5'end of the forward inner primer FIP (Table 1) for SARS -CoV-2 RT-LAMP colorimetric/fluorescence detection in vitro experiment.
  • FAM fluorescent labeling 5'-FAM-FIP
  • the N15(#1) and O117(#2) primer sets with 5'-FAM-FIP were used to detect the in vitro transcription RNA products of the N15 synthetic DNA fragment and the O117 synthetic DNA fragment, and the human ⁇ -actin primer set ( #3, SEQ ID No. 29-32) was used as a negative control, and water was used as a blank control (Blank).
  • the reaction product can be visually observed for the corresponding colorimetric results of each specific primer set, where a positive result is shown as the color changes from pink to yellow, and a negative result is shown as the color remains pink.
  • the fluorescent display of the positive result of the purified product under ultraviolet light is consistent with the colorimetric result.
  • the fluorescently labeled primer set of the present disclosure further overcomes the various uncertainties that may be encountered in the actual application of the RT-LAMP reaction, such as the influence of the difference of patient sample conditions on the change of pH, or the influence of the contrast color reading of various buffers, etc. , Through the dual display of colorimetry and fluorescence, the reliability of the diagnosis result is ensured.
  • this embodiment takes the N15 and O117 primer sets as an example, and the RT-LAMP reaction as an example, those skilled in the art will know that the same principle can also be similarly applied to other primer sets (such as N1, S17 primer sets). ), and similarly applied to LAMP reactions.
  • This example is a clinical experiment for the RT-LAMP detection of SARS-CoV-2 using the primer set of the present disclosure (taking N15 and O117 as examples).
  • VTM virus transport media
  • the virus inactivation step was carried out in accordance with the guidelines of the SARS-CoV-2 nucleic acid detection standard for clinical samples.
  • the swab sample was heated at 56°C for 30 minutes to make Its inactivation.
  • RNA extraction kits (Ningbo Haier Gene Technology Co., Ltd., Ningbo, China) were used to extract from swab samples RNA.
  • the RNA was eluted with water without RNase and DNase.
  • a commercially available 2019-nCoV RT-PCR kit (Shanghai ZJ Bio-Tech Co, Ltd., Shanghai, China) was used to determine whether 16 clinical samples were positive for the SARS-CoV-2 virus positive. According to the manufacturer's instructions, the ABI 7500 real-time PCR system (Thermo Fisher Scientific Inc., USA) was used for sequencing.
  • the reaction mixture (volume 25 ⁇ l) used is: 5 ⁇ l RNA template; 19 ⁇ l 2019-nCOV RT-PCR buffer; 1 ⁇ l RT-PCR enzyme mixture.
  • the thermal cycling conditions were: reverse transcription at 45°C for 10 minutes, initial PCR activation at 95°C for 3 minutes, and 45 cycles of 15s at 95°C and 30s at 58°C.
  • RT-LAMP analysis was performed on 16 clinical samples using pre-mixed test kits.
  • Each kit consists of three test tubes #1, #2, and #3, each of which contains O117, N15 and human ⁇ -actin primers (Table 1).
  • These kits were prepared in advance at Oxford Suzhou Centre For Advanced Research (OSCAR), and then shipped to Shenzhen Luohu People's Hospital in an ice box.
  • OSCAR Oxford Suzhou Centre For Advanced Research
  • RNA extracted from patient samples were added to #1, #2, and #3 tubes, respectively. Incubate the detection kit at 65°C for 30 minutes.
  • test tubes #1 and #2 of each kit turned yellow in all 8 positive samples, indicating that the target virus RNA was present in the samples, and in all 8 negative samples, test tubes #1 and #2 #2 all remain pink.
  • the No. 3 test tube of all test kits remained pink in both positive and negative samples, which indicates that human ⁇ -actin primers cannot detect ⁇ -actin gene transcripts in patient samples.
  • RT-qPCR has a Ct value of 38 for the Orf1ab gene, which indicates that the visual color change reading of RT-LAMP is highly sensitive.
  • Ct cycle threshold
  • ORF1ab orf1ab gene
  • N N gene
  • E E gene
  • IC internal standard, RNase P gene.
  • Example 7 RT-LAMP reaction experiment of one-step nucleic acid detection method
  • This example uses human cell lines and human primers as examples to test the RT-LAMP reaction example of the one-step nucleic acid detection method described in the present disclosure.
  • Those skilled in the art can know that by changing appropriate primers, enzymes, and reaction reagents, the method described in this embodiment can also be used to detect other different cells in various samples.
  • human cell line Human episomal iPSC line, product number A18945, ThermoFisher Scientific Ltd
  • the treatment groups in this experiment are:
  • HBSS (1) 0 cells + human ⁇ -actin primers; (2) 10 cells + human ⁇ -actin primers; (3) 50 cells + human ⁇ -actin primers; (4) 100 Cell + human ⁇ -actin primer; (5) hRNA + human ⁇ -actin primer; (6) water + human ⁇ -actin primer; (7) water (no primer); and
  • the human primers used in this example are the human ⁇ -actin primers used in the previous examples.
  • the reagents and RT-LAMP experimental procedures used are as described in Example 4, but the reaction time is 40 minutes.
  • the reaction system realizes cell lysis, RNA extraction, RNA reverse transcription and DNA amplification in one step, and the positive result can be observed with the naked eye under natural light, which has the characteristics of high efficiency and simplicity.

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Abstract

La présente invention concerne une amorce et un kit de test utilisés pour l'amplification génique du SARS-CoV-2 et, en combinaison avec des systèmes d'amplification génique comprenant LAMP ou RT-LAMP et analogues, ils peuvent identifier des patients atteints de COVID-19 ainsi que des porteurs asymptomatiques et déterminer si le virus du SARS-CoV-2 est présent dans des échantillons environnementaux, etc; des instruments compliqués sont inutiles et, facultativement, les résultats de test peuvent être lus à l'œil nu; ainsi l'invention est appropriée pour le criblage du virus du SARS-CoV-2 à grande échelle. La présente invention concerne également un procédé et une application de ladite amorce et dudit kit de test.
PCT/CN2021/083270 2020-03-27 2021-03-26 Amorce pour la détection du nouveau coronavirus du sars-cov-2, kit de test, procédé de détection et application correspondante WO2021190633A1 (fr)

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