WO2023159572A1 - Kit de détection, composition d'amorces et procédé de détection pour le nouveau coronavirus sars-cov-2 - Google Patents

Kit de détection, composition d'amorces et procédé de détection pour le nouveau coronavirus sars-cov-2 Download PDF

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WO2023159572A1
WO2023159572A1 PCT/CN2022/078307 CN2022078307W WO2023159572A1 WO 2023159572 A1 WO2023159572 A1 WO 2023159572A1 CN 2022078307 W CN2022078307 W CN 2022078307W WO 2023159572 A1 WO2023159572 A1 WO 2023159572A1
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primer
gene
cov
loop
orflab
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PCT/CN2022/078307
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Chinese (zh)
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颜菁
袁青
刘艳
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江苏汇先医药技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the invention belongs to the field of detection kits, and relates to a detection kit, a primer composition, and a detection method for novel coronavirus SARS-CoV-2, in particular to a detection reagent for detecting novel coronavirus SARS-CoV-2 based on a LAMP method box.
  • Novel coronavirus pneumonia is an acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2 or 2019-nCoV) infection, mainly lung lesions, clinical manifestations are fever , fatigue, dry cough as the main manifestations, nasal congestion, runny nose and other upper respiratory symptoms are rare. About half of the patients developed dyspnea more than a week later, and severe cases rapidly progressed to acute respiratory distress syndrome, septic shock, metabolic acidosis that was difficult to correct, and coagulation dysfunction.
  • SARS-CoV-2 or 2019-nCoV severe acute respiratory syndrome coronavirus type 2
  • the in vitro nucleic acid detection of the new coronavirus is mainly used to judge whether it is infected with the new coronavirus.
  • Fluorescence RT-PCR method which is widely used at present, although this method is widely used now, has certain deficiencies in detection accuracy and sensitivity, and requires large precision instruments and professional technicians to operate, which invisibly causes detection Due to the long time and high cost of detection, the detection of the new coronavirus can only be completed in professional genetic testing institutions or tertiary hospitals, and it cannot yet become an on-site instant detection method for the new coronavirus, which cannot effectively alleviate the current shortage of new coronavirus detection. question.
  • the object of the present invention is to provide a kind of detection kit and primer composition, detection method of novel coronavirus SARS-CoV-2, while its detection accuracy rate is higher, sensitivity is better and easy to operate, detection The time is shorter.
  • a detection kit for novel coronavirus SARS-CoV-2 includes the first primer set for the Orflab gene of the novel coronavirus SARS-CoV-2 and the primer set for the novel coronavirus SARS-CoV-2.
  • F3 5'-TGTTTTAAGCGGACACAATC-3', SEQ ID NO.1;
  • FIP 5'-AGCACTCTTAAGAAGTTGAATGTCTTGCTAAACACTGTCTTCATG-3', SEQ ID NO.3;
  • F3 5'-CGGCAGTCAAGCCTCTTC-3', SEQ ID NO.7;
  • FIP 5'-TCCCCTACTGCTGCCTGGAGCGTTCCTCATCACGTAGTCG-3', SEQ ID NO.9;
  • each primer is individually packaged. Dilute and mix before use. More preferably, the molar ratio of the primers Orflab-F3, Orflab-B3, Orflab-FIP, Orflab-BIP, Orflab-LF and Orflab-LB of the first primer set is preferably 1:1:8:8:4:4, It has a good amplification effect on the Orflab gene, obtains the lowest detection limit on the Orflab gene, and improves the accuracy of the detection result.
  • the molar ratio of the primers N-F3, N-B3, N-FIP, N-BIP and N-LF of the second primer set is preferably 1:1:8:8:4, which has better amplification of the N gene The effect is to obtain the lowest detection limit for the N gene and improve the accuracy of the detection results.
  • each primer of the first primer set forms the first primer mixture; each primer of the second primer set forms the second primer mixture.
  • the molar ratio of the outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF, and loop primer LB of the first primer set is 1:1 :8:8:4:4; in the second primer mixture, the molar ratio of the outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, and loop primer LB of the second primer set is 1 :1:8:8:4.
  • the detection kit also includes a combination of one or more of the following substances: dNTPs, MgCl 2 , 10 ⁇ Isothermo Buffer, enzyme, 20 ⁇ EvaGreen Dye, template and ddH 2 0.
  • the kit also includes a positive quality control product, and the positive quality control product includes an armored virus containing a target fragment of the Orflab gene and an armored virus containing a target fragment of the N gene.
  • the template includes an armored virus containing a target fragment of an Orflab gene and an armored virus containing a target fragment of an N gene.
  • the interpretation standard of the detection kit is: if the fluorescence curves for the Orflab gene and the N gene all have "S" shaped curves, it is judged as positive; if the fluorescence curves for the Orflab gene and the N gene If neither of the curves has an "S"-shaped curve, it is judged as negative; if one of the fluorescence curves for the Orflab gene and the N gene has an "S"-shaped curve and the other does not have an "S”-shaped curve, re-test, if still If one has an "S"-shaped curve and the other does not have an “S”-shaped curve, it is judged as positive.
  • a primer composition for a novel coronavirus SARS-CoV-2 comprising a first primer set directed at the Orflab gene of the novel coronavirus SARS-CoV-2 and directed against the novel coronavirus
  • the second primer set of the N gene of the virus SARS-CoV-2 includes the outer primer F3, outer primer B3, outer primer B3, Inner primer FIP, inner primer BIP, loop primer LF and loop primer LB
  • the second primer set includes outer primer F3 and outer primer B3 whose nucleotide sequences are sequentially shown in SEQ ID NO.7 ⁇ SEQ ID NO.11 , inner primer FIP, inner primer BIP, and loop primer LB.
  • a method for detecting the novel coronavirus SARS-CoV-2 for non-diagnostic and therapeutic purposes using the above-mentioned detection kit or the above-mentioned primer composition, after mixing with the sample, A loop-mediated isothermal amplification reaction was performed.
  • the procedure of the amplification reaction is to react at 60-65°C for 18-30 minutes. More preferably, the procedure of the amplification reaction is to react at 65°C for 20 minutes.
  • the first amplification system is used to detect the Orflab gene in the sample, wherein: the first amplification system includes the first primer set, dNTPs, MgCl 2 , 10 ⁇ Isothermo Buffer, enzyme, 20 ⁇ EvaGreen Dye, template and ddH 2 0. More preferably, the template comprises an armored virus containing a target segment of the Orflab gene.
  • the second amplification system is used to detect the N gene in the sample, wherein: the second amplification system includes the second primer set, dNTPs, MgCl 2 , 10 ⁇ Isothermo Buffer, enzyme, 20 ⁇ EvaGreen Dye, template and ddH 2 0. More preferably, the template comprises an armored virus containing a target segment of the N gene.
  • the concentration of the primer composition is 0.2-2 ⁇ M
  • the concentration of dNTPs is 1.0-2.0 mM
  • the concentration of MgCl 2 is 5-10 mM.
  • the molar ratio of the outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF and loop primer LB of the first primer set is 1:1:8:8:4:4,
  • the molar ratio of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, and the loop primer LB of the second primer set is 1:1:8:8:4.
  • samples are respectively added to the first amplification system and the second amplification system, and loop-mediated isothermal amplification is performed respectively, so as to simultaneously detect whether the samples contain the Orflab gene and the N gene.
  • the present invention adopts the above scheme, and has the following advantages compared with the prior art:
  • the detection kit, primer composition, and detection method of the novel coronavirus SARS-CoV-2 of the present invention are detected based on the loop-mediated isothermal amplification method, and are respectively aimed at 6 of the Orflab gene and the N gene of the SARS-CoV-2 virus.
  • Design 4 specific primers for each region add 2 or 1 loop primers, and perform amplification in a constant temperature environment of 60-65°C, with good sensitivity (the lowest detection limit can reach 10copies/mL and 50copies/mL) , the operation is simple, and the time is short; and it can detect the multi-gene loci of the SARS-CoV-2 virus at the same time, which improves the accuracy of the detection; and does not rely on expensive professional equipment, and can be operated by ordinary personnel without specialization , providing the possibility for on-site instant detection of SARS-CoV-2 virus.
  • Fig. 1 is the minimum detection limit experimental result figure of the SARS-CoV-2 virus Orflab gene target of the kit of embodiment 2;
  • Fig. 2 is the minimum detection limit experimental result graph of the SARS-CoV-2 virus N gene target of the kit of Example 2.
  • primers were synthesized by General Biosystems (Anhui) Co., Ltd.; dNTPs were purchased from Suo Laibao Biotechnology Co., Ltd.; nucleic acid dye 20 ⁇ EvaGreen Dye was purchased from biotium; new crown orf1ab and N gene pseudovirus Synthesized by Shanghai Kamin Biotechnology Co., Ltd.
  • This embodiment designs and screens the primers for detecting the new coronavirus SARS-CoV-2 based on RT-LAMP, specifically including the RT-LAMP primer set for detecting the Orflab gene of the new coronavirus SARS-CoV-2 and detecting the new coronavirus SARS-CoV-2
  • RT-LAMP primer set for the N gene the specific process is described below.
  • NCBI downloaded other common coronaviruses and used them to design RT-LAMP primer sets. Specifically: Viral HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV sequences, BioEdit and SnapGene sequence comparison analysis, to obtain specific Orflab fragments and N fragment sequences.
  • the armored virus containing the Orflab target fragment was serially diluted with ddH 2 0, and the copy numbers were 10 6 copies/mL, 10 5 copies/mL, 10 4 copies/mL, and 10 3 copies/mL.
  • the armored virus containing the N gene target fragment was serially diluted with ddH 2 0, and the copy numbers were 10 6 copies/mL, 10 5 copies/mL, 10 4 copies/mL, and 10 3 copies/mL.
  • Amplification reaction system amplification primer Mix 2.2 ⁇ L, dNTPs 1.0-2.0mM, MgCl2: 5-10mM, 10 ⁇ Isothermo Buffer 2.5 ⁇ L, enzyme Mix 0.2 ⁇ L, 20X EvaGreen Dye 1 ⁇ L, template 5 ⁇ L, add ddH20 to make up to 25 ⁇ L, the amplification primer reaction program is 65° C. for 20 min.
  • the template includes two types, the template for the amplification reaction system of the Orflab gene is an armored virus containing the target fragment of the Orflab gene, and the template for the amplification reaction system of the Orflab gene is an armored virus containing the target fragment of the N gene.
  • the primers with the best amplification effect of the Orf1ab gene are the primers of the second group, and the primers of the third group are the best with the amplification effect of the N gene.
  • Embodiment 2 detection kit
  • the detection kit includes: first primer set, second primer set, dNTPs, MgCl 2 , 10 ⁇ Isothermo Buffer, enzyme, 20 ⁇ EvaGreen Dye, ddH 2 0.
  • the first primer set is the second primer set in Table 1
  • the second primer set is the third primer set in Table 2, and each primer is packaged independently.
  • the kit also includes templates used as positive quality controls, including two armored viruses containing Orflab gene and N gene target fragments as templates, which were synthesized by Shanghai Kamin Biotechnology Co., Ltd.
  • amplification reaction system including: amplification primer Mix 2.2 ⁇ L, dNTPs 1.4 mM, MgCl 2 8 mM, 10 ⁇ Isothermo Buffer 2.5 ⁇ L, enzyme Mix 0.2 ⁇ L, 20X EvaGreen Dye 1 ⁇ L, template 5 ⁇ L, add ddH20 Make up to 25 ⁇ L.
  • the amplification primer Mix in the first amplification reaction system is a mixture of primers F3, B3, FIP, BIP, LF and LB for the Orflab gene
  • the template is a mixture containing the Orflab gene target
  • the amplification primer Mix in the second amplification reaction system is a mixture of primers F3, B3, FIP, BIP, LF and LB for the N gene
  • the template is the armored virus containing the N gene target fragment.
  • the molar ratio of the primers Orflab-F3, Orflab-B3, Orflab-FIP, Orf1ab-BIP, Orflab-LF and Orflab-LB is preferably 1: 1:8:8:4:4.
  • the molar ratio of the primers N-F3, N-B3, N-FIP, N-BIP and N-LF is preferably 1:1:8: 8:4.
  • the primer mixture was prepared according to the molar ratio of the above primers.
  • the method of using the kit is as follows:
  • Sample processing Take a sample (such as a throat swab or oral swab sample) and transfer it to a centrifuge tube containing the sample lysate, shake and mix well, and wait for inspection;
  • a sample such as a throat swab or oral swab sample
  • step (1) Add the first amplification system (20 ⁇ L) and the liquid (5 ⁇ L) in step (1) to test tube 1, add the second amplification system (20 ⁇ L) and the liquid in step (1) to test tube 2 Liquid (5 ⁇ L), react in a nucleic acid detector at 65°C for 20 minutes;
  • an "S" curve indicates that the test sample contains the new coronavirus.
  • the two targets of the Orflab gene of the new coronavirus are consistent, it will be judged according to the consistent result; if one of the two targets is positive and the other is negative, the test will be repeated, and if the test result is still one negative and one positive, then it will be judged as positive. determination.
  • the armored virus containing the Orflab target fragment and the armored virus containing the N gene target fragment were serially diluted with ddH 2 0, and the copy numbers were 10 5 copies/mL, 10 4 copies/mL, 10 3 copies/mL, 100 copies/mL, 50copies/mL, 10copies/mL.
  • Use the kit of Example 2 to detect the Orflab target template according to the method described in Example 2 use the kit of Example 2 to detect the N gene target template according to the method described in Example 2, and determine the minimum detection method for positive nucleic acids quantity.
  • the minimum detection limit of the nucleic acid of the Orflab gene target of the new coronavirus SARS-CoV-2 is 10copies/mL; as shown in Figure 2, the minimum detection limit of the nucleic acid of the N gene target of the new coronavirus SARS-CoV-2 is 50copies /mL.
  • Influenza A virus H1N1
  • influenza B virus Yamagata
  • respiratory syncytial virus type A
  • adenovirus type 2
  • parainfluenza virus type 1
  • Middle East Respiratory Syndrome Coronavirus MERSr-CoV
  • Human Coronavirus HCoV-229E Human Coronavirus HCoV-HK U1, Human Coronavirus HCoV-NL63, Human Coronavirus HCoV-0C43
  • Influenza A Virus (H3N2) 13 viruses Influenza A virus (H5N1) and Influenza A virus (H7N9)
  • RNA virus genome extraction kit of Suleibao Biotechnology Co., Ltd. respectively.
  • Example 2 The kits in Example 2 were used to detect the above 13 viral nucleic acids, the Orflab gene target and N gene target templates were used as positive controls, and ddH 2 0 was used as a negative control.

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Abstract

La présente invention propose un kit de détection, une composition d'amorces et un procédé de détection pour le nouveau coronavirus SARS-CoV-2. Le kit de détection pour le nouveau coronavirus SARS-CoV-2 comprend un premier ensemble d'amorces pour un gène Orflab du nouveau coronavirus SARS-CoV-2 et un deuxième ensemble d'amorces pour un gène N du nouveau coronavirus SARS-CoV-2 ; le premier ensemble d'amorces comprend l'amorce extérieure F3, l'amorce extérieure B3, l'amorce intérieure FIP, l'amorce intérieure BIP, l'amorce en boucle LF et l'amorce en boucle LB ayant des séquences de nucléotides consécutives telles qu'indiquées dans SEQ ID NO : 1 à SEQ ID NO : 6 ; et le deuxième ensemble d'amorces comprend l'amorce extérieure F3, l'amorce extérieure B3, l'amorce intérieure FIP, l'amorce intérieure BIP et l'amorce de boucle LB ayant des séquences de nucléotides séquentiellement comme indiqué dans SEQ ID NO : 7 à SEQ ID NO : 11. Le kit de détection, la composition d'amorce et le procédé de détection du nouveau coronavirus SARS-CoV-2 de la présente invention présentent une grande précision de détection, une bonne sensibilité, un fonctionnement pratique et un temps de détection court.
PCT/CN2022/078307 2022-02-28 2022-02-28 Kit de détection, composition d'amorces et procédé de détection pour le nouveau coronavirus sars-cov-2 WO2023159572A1 (fr)

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Cited By (1)

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CN117070673A (zh) * 2023-09-27 2023-11-17 广州动物园 用于穿山甲α冠状病毒的LAMP检测引物组及其用途

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CN114561491A (zh) * 2022-02-28 2022-05-31 江苏汇先医药技术有限公司 新型冠状病毒SARS-CoV-2的检测试剂盒及引物组合物、检测方法

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CN111020064A (zh) * 2020-03-10 2020-04-17 中山大学达安基因股份有限公司 新型冠状病毒ORF1ab基因核酸检测试剂盒
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CN114561491A (zh) * 2022-02-28 2022-05-31 江苏汇先医药技术有限公司 新型冠状病毒SARS-CoV-2的检测试剂盒及引物组合物、检测方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117070673A (zh) * 2023-09-27 2023-11-17 广州动物园 用于穿山甲α冠状病毒的LAMP检测引物组及其用途
CN117070673B (zh) * 2023-09-27 2024-02-06 广州动物园 用于穿山甲α冠状病毒的LAMP检测引物组及其用途

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