WO2021211012A1 - Système crispr-cas pour révéler une gène de résistance aux antibiotiques - Google Patents

Système crispr-cas pour révéler une gène de résistance aux antibiotiques Download PDF

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WO2021211012A1
WO2021211012A1 PCT/RU2020/050305 RU2020050305W WO2021211012A1 WO 2021211012 A1 WO2021211012 A1 WO 2021211012A1 RU 2020050305 W RU2020050305 W RU 2020050305W WO 2021211012 A1 WO2021211012 A1 WO 2021211012A1
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blavim
antibiotic resistance
pseudomonas aeruginosa
resistance gene
crispr
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Василий Геннадьевич АКИМКИН
Александр Игоревич ТЮМЕНЦЕВ
Марина Алексеевна ТЮМЕНЦЕВА
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Федеральное Бюджетное Учреждение Науки "Центральный Научно-Исследовательский Институт Эпидемиологии" Федеральной Службы По Надзору В Сфере Защиты Прав Потребителей И Благополучия Человека
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Definitions

  • the invention relates to the field of genetic engineering and biotechnology, namely to the number of means - guide RNAs that can be used in CRISPR-Casl2 systems as part of ribonucleoprotein complexes for the detection (detection, detection) of the antibiotic resistance gene blaVIM-2 (metal-beta-lactamase class VIM-2) Pseudomonas aeruginosa.
  • blaVIM-2 metal-beta-lactamase class VIM-2
  • the invention allows in vitro detection of single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.
  • the guide RNAs described in this application can be used to detect the blaVIM-2 antibiotic resistance gene
  • Amplification in this case can be carried out in various ways, including polymerase chain reaction (PCR); loop isothermal amplification (LAMP); helicase-dependent amplification (HDA); recombinase-mediated amplification (RPA); strand displacement amplification (SDA); nucleic acid sequence based amplification (NASBA); transcription-mediated amplification (TMA); diving enzyme-mediated amplification (NEAR); circular amplification (RCA) and many other types of amplification.
  • PCR polymerase chain reaction
  • LAMP loop isothermal amplification
  • HDA helicase-dependent amplification
  • RPA recombinase-mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • TMA transcription-mediated amplification
  • NEAR diving enzyme-mediated amplification
  • RCA circular amplification
  • the guide RNAs described in this application can be used to develop highly sensitive and high-tech new generation diagnostic systems based on CRISPR technologies to combat the spread of antibiotic-resistant bacterial pathogens.
  • the proposed platform combines Casl2a nuclease, its guide RNA, HPV nucleic acid specific, and fluorescent reporter molecule.
  • DETECTR technology is used to detect a target DNA target after preliminary amplification (Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA. CRISPR-Casl2a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018 Apr 27; 360 (6387): 436-439).
  • CRISPR / CAS system An equally important application of the CRISPR / CAS system is the identification of bacterial pathogens and the detection of specific bacterial genes. So, for example, using the SHERLOCK platform (Specific High Sensitivity Enzymatic Reporter UnLOCKing - Specific
  • the proposed analysis only in the future can be applied to samples with a low concentration of DNA - the proposed method describes the analysis with samples containing about 10 8 copies of plasmid DNA carrying antibiotic resistance genes (60 yg DNA, plasmid DNA 67 kb in size). - 220 kb), which is its significant drawback. Also, the disadvantages of the described solution are the need to use expensive high-tech equipment (specialized nanofluid biochips, an inverted fluorescence microscope with a magnification of at least 100x), as well as the need for complex analysis of the data obtained using specialized software.
  • CRISPR / CAS based technology is a multifunctional, error resistant DNA detection technology suitable for rapid set-up diagnoses, including infectious diseases, genotyping of infectious agents and identification of genes for antibiotic resistance of bacterial pathogens.
  • the invention relates to new means - guide RNAs, which can be used in CRISPR-Casl2 systems for ultrasensitive detection, identification, detection or detection of the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa in biological samples.
  • the technical objective of the proposed invention is the development of new means - guide RNAs that can be used in CRISPR-Casl2 systems with Casl2 proteins, for example, LbCpfl from Lachnospiraceae, for ultrasensitive detection of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.
  • an unexpected technical result is achieved - the possibility of ultrasensitive detection of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa to single copies of DNA in one reaction.
  • the invention provides increasing the efficiency of detecting the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa from 1-5x10 5 to 2-3x10 2 copies / ml.
  • RNA hairpin which is recognized by the RNA-directed DNA endonuclease LbCpfl from Lachnospiraceae, ensuring the detection of single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.
  • RNA-directed DNA endonuclease LbCpfl from Lachnospiraceae, obtained according to the method developed by the authors earlier (invention under RF patent N ° 2707542, priority date 03/28/2019, published 11/27/2019), to create ribonucleoprotein complexes (RPK) of the CRISPR / CAS, suitable for the detection of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa at ultra-low concentrations (single copies).
  • RPK ribonucleoprotein complexes
  • a CRISPR / CAS targeting RNA molecule has been proposed that can bind to target highly conserved regions of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa.
  • the nucleotide sequence of the guide RNA disclosed herein is selected from SEQ ID NO: 1-5.
  • the guide RNA molecule contains an RNA hairpin, which is recognized by the RNA guided DNA endonuclease LbCpfl from Lachno spiraceae.
  • the guide RNA molecule ensures the detection of single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.
  • the guide RNAs of the present invention correspond to highly conserved fragments of the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene. Most preferred are guide RNAs recognized by the RNA-directed DNA endonuclease LbCpfl from Lachnospiraceae, characterized by having or containing a nucleotide sequence selected from:
  • ribonucleoprotein complexes consisting of at least one guide RNA and RNA-guided DNA endonuclease of the CRISPR / CAS LbCpfl system from Lachnospiraceae, suitable for use for detecting the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa in ultra-low concentrations copies).
  • Ribonucleoprotein complex of the CRISPR / CAS system for detecting the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa formed from RNA-directed DNA endonuclease LbCpfl from Lachnospiraceae, and at least one guide RNA.
  • RPK The proposed ribonucleoprotein complex of the CRISPR / CAS system (hereinafter referred to as RPK) is suitable for detecting single copies of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa.
  • PKK preparations are solutions containing a guide RNA selected from SEQ ID NO: 1-5, combined with a CRISPR / CAS protein (LbCpfl from Lachnospiraceae) or freeze-dried PKK.
  • the resulting guide RNAs can be used as part of a kit for detecting the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa, with instructions for use.
  • the kit for detecting the antibiotic resistance gene blaVIM-2 from Pseudomonas aeruginosa may contain a ribonucleoprotein complex and instructions for use.
  • At least one guide RNA in the kit can be complexed with a CRISPR / CAS protein (LbCpfl from Lachnospiraceae) in one container or separately in different containers.
  • a CRISPR / CAS protein LbCpfl from Lachnospiraceae
  • the kit may additionally include components for pre-amplifying highly conserved fragments of the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene, including one or more specific oligonucleotides selected from SEQ ID NO: 6 and SEQ ID NO: 7.
  • oligonucleotides for carrying out preliminary amplification of a fragment of the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene correspond to the highly conserved region of the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene.
  • Most preferred are oligonucleotides characterized by having or containing a nucleotide sequence selected from:
  • a biological sample can be a sample of blood, serum or blood plasma, blood cells, saliva, sputum, lymphoid tissues, tissues of hematopoietic organs and other biological materials from a patient, which can be used to analyze for the presence of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.
  • FIG. 1 Visualization of a fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa from 37 to 447 and. O. (411 i.p.) after preliminary amplification using agarose gel electrophoresis, where numbers 1-8 indicate:
  • FIG. 2 Visualization of PCR products encoding guide RNAs specific to the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa using agarose gel electrophoresis, where numbers 1-5 indicate:
  • M - molecular weight standards from bottom to top 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 3000 base pairs (GeneRuler 100 bp Plus, Thermo Fisher Scientific, USA).
  • FIG. 3 Real-time fluorescence profile for a pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with a ribonucleoportein complex containing sgRNA blaVIM-2 93 guide RNA and LbCpfl protein.
  • FIG. 4 Real-time fluorescence profile for a pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with a ribonucleoportein complex containing sgRNA blaVIM-2> 95 guide RNA and LbCpfl protein.
  • FIG. 5 Real-time fluorescence profile for a pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with a ribonucleoportein complex containing sgRNA blaVIM-2 207 guide RNA and LbCpfl protein.
  • FIG. 6 Real-time fluorescence profile for a pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with a ribonucleoportein complex containing sgRNA blaVIM-2 285 guide RNA and LbCpfl protein.
  • FIG. 7 Real-time fluorescence profile for a pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with a ribonucleoportein complex containing a guide RNA sgRNA blaVIM-2> 366 and LbCpfl protein.
  • FIG. 8 Endpoint fluorescence values (30 assay cycle, 30 minutes) for pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with ribonucleoportein complex containing guide RNA sgRNA blaVIM-2 N ° 93, sgRNA blaVIM-2 N ° 95, sgRNA blaVIM-2 -2 JVb207, sgRNA blaVIM-2 JVb285 and sgRNA blaVIM-2 Zh366.
  • FIG. 9 Endpoint fluorescence values (30 assay cycle, 30 minutes) for Pseudomonas aeruginosa blaVIM-2 targets pre-amplified from clinical samples treated with ribonucleoportein complex containing sgRNA blaVIM-2 guide RNA N ° 93.
  • FIG. 10 Endpoint fluorescence values (30 analysis cycle, 30 minutes) for Pseudomonas aeruginosa blaVIM-2 targets pre-amplified from clinical samples treated with ribonucleoportein complex containing the blaVIM-2 sgRNA guide RNA> 95.
  • FIG. 11 Endpoint fluorescence values (30 assay cycle, 30 minutes) for Pseudomonas aeruginosa blaVIM-2 targets pre-amplified from clinical samples treated with ribonucleoportein complex containing sgRNA blaVIM-2 JVb207 guide RNA.
  • FIG. 12 Endpoint fluorescence values (30 analysis cycle, 30 minutes) for Pseudomonas aeruginosa blaVIM-2 targets pre-amplified from clinical samples treated with ribonucleoportein complex containing sgRNA blaVIM-2 * G ° 285 guide RNA.
  • FIG. 13 Endpoint fluorescence values (30 analysis cycle, 30 minutes) for Pseudomonas aeruginosa blaVIM-2 targets pre-amplified from clinical samples, treated with ribonucleoportein complex containing guide RNA sgRNA blaVIM-2 Zbb.
  • Example 1 Selection of target sequences in the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa to create guide RNAs.
  • To select target sequences in the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene to create guide RNAs modern algorithms for in silico analysis of nucleotide sequences and publicly available programs were used, including Benchling (https://www.benchling.com/molecularbiology /).
  • Benchling https://www.benchling.com/molecularbiology /.
  • a list of regions in the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa with a theoretically calculated probability of their cleavage in highly conserved regions was compiled (Table 1).
  • Table 1 List of regions in the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa with the theoretically calculated probability of their cleavage by LbCpfl from Lachnospiraceae.
  • the guide RNAs that specifically recognize the highly conserved antibiotic resistance site of blaVIM-2 of Pseudomonas aeruginosa are represented by the unique sequences SEQ III NO: 1-5.
  • Example 2 Preparation of material for the detection of the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa by the method of preliminary amplification.
  • Preparation of material for the detection of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa is carried out by the method of preliminary amplification.
  • a model matrix of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa of a biological sample plasmid DNA pGEM-T-blaVIM-2 containing a fragment of the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa from 37 to 447 bp. (size 411 bp).
  • a PCR product encoding a fragment of the antibiotic resistance blaVIM-2 of Pseudomonas aeruginosa is obtained in an amplification reaction using PCR mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides For blaVIM-2 and Rev blaVIM-2 (GenTerra, Russia) ...
  • the size of the amplified blaVIM-2 fragment is 411 base pairs.
  • the obtained fragments of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa are visualized using agarose gel electrophoresis (Fig. 1).
  • the material prepared by the described method is used for experiments on the detection of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa using ribonucleoprotein complexes LbCpfl from Lachnospiraceae containing guide RNA sgRNA blaVIM-2 * G ° 93, sgRNA blaVIM-2 blaVIM-2 AN 5, sgRNA blaVIM-2 JVb285 and sgRNA blaVIM-2 JV “366, without preliminary purification.
  • Example 3 Obtaining guide RNAs and creation of ribonucleoprotein complexes for the detection of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.
  • the PCR product encoding the guide RNA sgRNA blaVIM-2 N ° 93 is obtained in an amplification reaction using PCR mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and sgRNA-93-Rev (GenTerra, Russia) ...
  • the size of the amplified fragment encoding sgRNA blaVIM-2 N ° 93 is 62 bp.
  • the PCR product encoding the guide RNA sgRNA blaVIM-2 N ° 95 is obtained in an amplification reaction using PCR mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and sgRNA-95-Rev (GenTerra, Russia) ...
  • the size of the amplified fragment encoding sgRNA blaVIM-2 N ° 95 is 62 bp.
  • the PCR product encoding the guide RNA sgRNA blaVIM-2 N ° 207 is obtained in an amplification reaction using PCR mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and sgRNA-207-Rev (GenTerra, Russia) ...
  • the size of the amplified fragment encoding sgRNA blaVIM-2 N ° 207 is 62 base pairs.
  • the PCR product encoding the guide RNA sgRNA blaVIM-2 N ° 285 is obtained in an amplification reaction using PCR mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and sgRNA-285-Rev (GenTerra, Russia) ...
  • the size of the amplified fragment encoding sgRNA blaVIM-2 N ° 285 is 62 base pairs.
  • the PCR product encoding the guide RNA sgRNA blaVIM-2 N ° 366 is obtained in an amplification reaction using PCR mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and sgRNA-366-Rev (GenTerra, Russia) ...
  • the size the amplified fragment encoding sgRNA blaVIM-2 N ° 366 is 62 base pairs.
  • Amplification temperature profile for obtaining PCR products encoding guide RNAs :
  • PCR products encoding guide RNAs specific to a fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa were visualized by agarose gel electrophoresis (Fig. 2).
  • Pseudomonas aeruginosa is carried out using a commercially available ISOLATE II PCR and Gel Kit (BioLine, USA) according to the manufacturer's instructions. Purified PCR products encoding guide RNAs specific to the blaVIM-2 antibiotic resistance gene fragment
  • Pseudomonas aeruginosa is used as a template for the synthesis of guide RNAs.
  • the synthesis of guide RNAs specific to a fragment of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa is carried out by in vitro transcription using commercially available reagent kits (HiScribe TM T7 High Yield RNA Synthesis Kit, NEB, USA) according to the manufacturer's instructions.
  • the products of the in vitro transcription reaction are reprecipitated from the reaction mixture by adding sodium chloride to a final concentration of 400 tM and an equal volume of isopropyl alcohol.
  • Such modifications of the manufacturer's protocol, introduced by the authors, allow increasing the yield of the reaction product and obtaining the desired concentration of the final guide RNA preparation.
  • the guide RNA preparation (250 ng) is heated at 90 ° C for 5 minutes and allowed to cool slowly to room temperature (incubation at room temperature for at least 10 minutes). This heating is necessary for the formation of the correct conformation of the hairpin contained in the guide RNA. Many manufacturers of commercial CAS protein preparations skip this step when preparing the ribonucleoprotein complex.
  • ribonucleoprotein complex 250 ng of the LbCpfl CAS protein from Lachnospiraceae and the prepared guide RNA are mixed and incubated for 15 minutes at room temperature.
  • the ribonucleoprotein complex obtained in this way is ready for the detection of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.
  • Example 4 Detection of single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa using ribonucleoprotein complexes CRISPR / CAS using a model matrix as an example.
  • the pre-amplified material obtained by the method described in Example 2 is used as a template for detecting the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa using ribonucleoprotein complexes CRISPR / CAS obtained by the method described in Example 3.
  • reaction mixture is prepared containing the following components:
  • reaction mixtures containing all the necessary components are placed in a DTPrime 5 thermocycler (DNA-Technology, Russia) and the following reaction parameters are set:
  • CRISPR / CAS ribonucleoprotein complexes are capable of detecting single copies of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa.
  • Typical analysis results are shown using real-time fluorescence profiles for a pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with ribonucleoportein complexes containing guide RNAs sgRNA blaVIM-2 JVb93, sgRNA blaVIM-2 JVb95, sg7-2 sgRNA blRNA blaVIM-2 J ⁇ ° 285 and sgRNA blaVIM-2 366 and LbCpfl protein, in FIG.
  • ribonucleoprotein complexes CRISPR / CAS formed on the basis of LbCpfl from Lachnospiraceae and guide RNAs, reveal single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa with different efficiencies, and they can be arranged in the following order of decreasing activity: sgRNA blaVIM 207> sgRNA blaVIM-2 N ° 93> sgRNA blaVIM-2 F 66> sgRNA blaVIM-2 L ° 285> sgRNA blaVIM-2> 95 (Fig. 8).
  • Example 5 Detection of single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa using ribonucleoprotein complexes CRISPR / CAS on a limited panel of clinical samples.
  • the developed guide RNAs were tested on a limited panel of clinical samples (10 pcs.) Containing
  • PCR products encoding a fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa are obtained in an amplification reaction using PCR-mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) as described in Example 2, using the described temperature profile and duration of the amplification reaction.
  • the material obtained in this way is used as a template for the detection of single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa using ribonucleoprotein complexes CRISPR / CAS obtained by the method described in Example 3. Detection of single copies of the antibiotic resistance gene blaVIM-
  • CRISPR / CAS ribonucleoprotein complexes are capable of detecting single copies (1.5 copies / reaction) of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa in DNA preparations isolated from clinical samples.
  • the signal value exceeded the value of "noise” (nonspecific fluorescence of the control sample containing no target) by three times, and by the 5-26 cycle (5-26 minutes) of the analysis - more than 5 times (wide range and difference in cycles are due to differences in guide RNAs used in the analysis, Table 5).
  • Typical assay results are exemplified by fluorescence endpoint values (30 assay cycles, 30 minutes) for pre-amplified blaVIM-2 Pseudomonas aeruginosa targets (10 independent clinical samples) treated with ribonucleoportein complexes containing guide RNA sgRNA blaVIM-2 JVb93, sgRNA blaVIM-2 JVb95, sgRNA blaVIM-2 JVb207, sgRNA blaVIM-2 JVT2285 and sgRNA blaVIM-2 K2Cp66, and protein Lb. 9, Fig. 10, Fig. 11, Fig. 12 and FIG. 13 respectively.
  • the efficiency of detecting single copies of the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene contained in DNA preparations isolated from clinical samples using various guide RNAs in the CRISPR / CAS ribonucleoprotein complexes formed on the basis of LbCpfl from Lachnospiraceae can be presented in the following descending order : sgRNA blaVIM-2 207> sgRNA blaVIM-2 93> sgRNA blaVIM-2 J ⁇ ° 285> sgRNA blaVIM-2 K2Z66> sgRNA blaVIM-2 95 (Table 5).
  • the developed guide RNAs allow ultrasensitive detection of single copies of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa in clinical samples after preliminary amplification in the CRISPR / CAS ribonucleoprotein complexes.

Abstract

L'invention se rapporte au domaine du génie génétique et des biotechnologies et concerne notamment un certain nombre de moyens - ARN guides, des complexes ribonucléoprotéiniques de système CRISPR-CAS comprenant des ARN guides, et des kits comprenant des complexes ribonucléoprotéiniques de système CRISPR-CAS et des oligonucléotides spécifiques pour l'amplification préalable d'une section fortement conservatrice du gène de résistance aux antibiotiques blaVIM-2 Pseudomonas aeruginosa. L'invention permet de révéler efficacement le gène de résistance aux antibiotiques blaVIM-2 Pseudomonas aeruginosa après avoir effectué une amplification spécifique d'un fragment de ce gène. Les ARN guides sont représentés par les séquences SEQ ID NO: 1-5, et les oligonucléotides spécifiques pour l'amplification préalable sont représentés par les séquences SEQ ID NO: 6 et SEQ ID NO: 7 L'invention permet de révéler des copies uniques du gène de résistance aux antibiotiques blaVIM-2 Pseudomonas aeruginosa.
PCT/RU2020/050305 2020-04-15 2020-10-28 Système crispr-cas pour révéler une gène de résistance aux antibiotiques WO2021211012A1 (fr)

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WO2016205711A1 (fr) * 2015-06-18 2016-12-22 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes

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RU2707542C1 (ru) * 2019-03-28 2019-11-27 Федеральное бюджетное учреждение науки "Центральный научно-исследовательский институт эпидемиологии" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФБУН ЦНИИ Эпидемиологии Роспотребнадзора) Способ получения препарата рекомбинантной нуклеазы CAS, по существу, свободного от бактериальных эндотоксинов, полученный данным способом препарат и содержащий его набор для использования в системе CRISPR/Cas

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WO2016205711A1 (fr) * 2015-06-18 2016-12-22 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes

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