WO2020101194A1 - Ensemble d'amorces pour une réaction d'amplification isothermique médiée par boucle pour diagnostiquer des entérobactéries productrices de carbapénèmase, et utilisation correspondante - Google Patents

Ensemble d'amorces pour une réaction d'amplification isothermique médiée par boucle pour diagnostiquer des entérobactéries productrices de carbapénèmase, et utilisation correspondante Download PDF

Info

Publication number
WO2020101194A1
WO2020101194A1 PCT/KR2019/013707 KR2019013707W WO2020101194A1 WO 2020101194 A1 WO2020101194 A1 WO 2020101194A1 KR 2019013707 W KR2019013707 W KR 2019013707W WO 2020101194 A1 WO2020101194 A1 WO 2020101194A1
Authority
WO
WIPO (PCT)
Prior art keywords
primer
seq
loop
carbapenemase
primer set
Prior art date
Application number
PCT/KR2019/013707
Other languages
English (en)
Korean (ko)
Inventor
강래형
백순영
박연준
Original Assignee
가톨릭대학교산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 가톨릭대학교산학협력단 filed Critical 가톨릭대학교산학협력단
Publication of WO2020101194A1 publication Critical patent/WO2020101194A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • 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
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/101—Temperature
    • 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
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/113—PCR
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a primer set for loop-mediated isothermal amplification reaction for the diagnosis of carbapenemase-producing intestinal bacteria and uses thereof, and more specifically, loop-mediated isotherm for IMP, VIM, KPC, and NDM known as carbapenemase-generating genes.
  • Carbapenem-based antibiotics are widely used antibiotics for the treatment of Gram-negative bacterial infections.
  • Antibiotics of the carbapenem family include imipenem, meropenem, Invanz, ertapenem, and doripenem.
  • the carbapenem-based antibiotics are known to be the most powerful antibiotics, and are mainly used for patients with severe infections.
  • Carbapenem-based antibiotics have also become a major social problem as resistant strains have been reported worldwide.
  • the intestinal bacteria resistant to these carbapenem-resistant antibiotics are called carbapenem resistant enterobacteriaceae (CRE).
  • CRE carbapenem resistant enterobacteriaceae
  • ⁇ -lactamase to neutralize the carbapenem-based antibiotics
  • ⁇ -lactamase inactivates antibiotics. (Enzyme inactivation) causing antibiotic resistance.
  • Bacteria that are resistant to the antibiotics of the carbapenem family are E. coli and pneumococcal ( Klebsiella pneumoniae ), as well as several strains that have genes that can produce ⁇ -lactamase enzymes. It is known to have.
  • genes generating ⁇ -lactamase genes such as KPC, IMP, GES, SIM, and VIM, as well as NDM genes, have been reported. Genes that generate such ⁇ -lactamase enzymes exist in several genotypes. The most representative of these, NDM (New Delhi metallo beta lactamase), was named in 2008 after the Swedish patient was confirmed to be infected during surgery in New Delhi, India. Bacteria carrying the NDM gene are known to be resistant to almost all existing antibiotics, and are fatal, and infection experts are concerned because they are likely to spread worldwide in recent years in India and Pakistan, as well as in the United States and Japan. As such, the risk of multi-drug resistant bacteria including CRE has emerged worldwide, and in Korea, in September 2010, 'Cabapenem-resistant enteric bacteria (CRE)' among multi-drug resistant bacteria was urgently designated as a legal infectious disease.
  • CRE Cabapenem-resistant enteric bacteria
  • CPE carbapenemase producing enterobacteriaceae
  • CPE carbapenemase producing enterobacteriaceae
  • a total of 59 CPEs were identified by 2012, and in July 2013, as new CPEs that were not reported in Korea were identified, epidemiological investigations were conducted at the Center for Disease Control and confirmed that 63 hospitals and 63 patients were infected with CPE. Became.
  • CPE is a type of CRE having a gene that generates an enzyme capable of directly degrading antibiotics among CREs, and has the potential to transmit resistance to other strains by a plasmid.
  • Representative CPEs include Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo- ⁇ -lactamase-1 (NDM-1).
  • the methods of testing for the presence of carbapenem-resistant enterococci are disc diffusion method, Modified Hodge Test (MHT) method, Carbapenemase Nordmann-poirel test, Carbapenemase inhibition test, PCR amplification ⁇ Electrophoresis, DNA sequencing methods are used.
  • MHT Modified Hodge Test
  • Carbapenemase Nordmann-poirel test Carbapenemase inhibition test
  • PCR amplification ⁇ Electrophoresis DNA sequencing methods are used.
  • a microarray-type DNA chip is a method that can simultaneously test tens to tens of thousands of genes on a single slide, or an endpoint analysis that simply identifies the genotype by hybridizing the product after the PCR amplification reaction to a DNA chip integrated with a probe. (end-point analysis) method.
  • the PCR amplification process is a linear step in which the amount of DNA is arithmeticly increased as the amplification rate begins to decrease as the components required for PCR amplification gradually deplete after exponential phases with a constant DNA amplification rate and high reproducibility. The PCR amplification stops after (linear phases) and proceeds to the plateau phases with the lowest reproducibility.
  • the biggest problem of the microarray DNA chip method is that it is difficult to design primers and probes for PCR amplification that can detect multiple target genes while satisfying both specificity and sensitivity, and it is difficult to design a fluorescence intensity cutoff value for positive and negative determination. There is a problem that is difficult to determine.
  • the specificity and sensitivity of the carbapenem-resistant enterococci are quickly and accurately determined based on the quantified data, and the specificity and sensitivity are high, instead of the conventional disk diffusion method, DNA sequencing method, or multiplex microarray DNA chip.
  • the development of a method that can be done is an urgent situation.
  • Carbapenemase-producing intestinal bacteria are currently diagnosed or diagnosed within 2 hours using Xpert (Cepheid) using real-time PCR, but in the case of Expert, expensive equipment and reagents are required. The disadvantage is that it takes two hours.
  • the loop-mediated isothermal amplification (LAMP) method is a simple and fast isothermal amplification method using a Bst DNA polymerase having a strand displacement DNA synthesis function.
  • LAMP loop-mediated isothermal amplification
  • a primer set for the LAMP reaction was prepared, and the primer set of the present invention was equipped with equipment such as a simple heating block. It was confirmed that the test could be performed within 1 hour, and it was confirmed that the intestinal bacteria producing carbapenemase were effectively diagnosed, and the present invention was completed.
  • An object of the present invention is to provide a set of primers for loop-mediated isothermal amplification reaction for the diagnosis of carbapenemase-producing intestinal bacteria.
  • Another object of the present invention is to provide a carbapenemase-producing enterococcal bacteria diagnostic kit comprising the primer set for the loop-mediated isothermal amplification reaction.
  • Another object of the present invention is to provide a method for diagnosing intestinal bacteria producing carbapenemase using the primer set for the loop-mediated isothermal amplification reaction.
  • Primer set for IMP gene detection consisting of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 6;
  • KPC gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 29 to SEQ ID NO: 34;
  • NDM gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 43 to SEQ ID NO: 48; Provided is a primer set for loop-mediated isothermal amplification (LAMP) for detecting carbapenemase producing enterobacteriaceae (CPE), which is composed of any one or more primer sets.
  • LAMP loop-mediated isothermal amplification
  • CPE carbapenemase producing enterobacteriaceae
  • the primer set for IMP gene detection is a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, a forward inner primer of SEQ ID NO: 3 (FIP), SEQ ID NO: 4 It consists of a reverse inner primer (Back inner pimer; BIP), a forward loop primer of SEQ ID NO: 5 (LoopF) and a reverse loop primer of SEQ ID NO: 6 (LoopB),
  • the primer set for VIM gene detection includes a forward primer of SEQ ID NO: 15, a reverse primer of SEQ ID NO: 16, a forward inner primer of SEQ ID NO: 17 (FIP), and a reverse inner primer of SEQ ID NO: 18 (Back inner pimer; BIP) ), A forward loop primer of SEQ ID NO: 19 (LoopF) and a reverse loop primer of SEQ ID NO: 20 (LoopB),
  • the KPC gene detection primer set includes a forward primer of SEQ ID NO: 29, a reverse primer of SEQ ID NO: 30, a forward inner primer of SEQ ID NO: 31 (FIP), and a reverse inner primer of SEQ ID NO: 32 (Back inner pimer; BIP) ), A forward loop primer of SEQ ID NO: 33 (LoopF) and a reverse loop primer of SEQ ID NO: 34 (LoopB),
  • the NDM gene detection primer set includes a forward primer of SEQ ID NO: 43, a reverse primer of SEQ ID NO: 44, a forward inner primer of SEQ ID NO: 45 (FIP), and a reverse inner primer of SEQ ID NO: 46 (Back inner pimer; BIP). ), The forward loop primer of SEQ ID NO: 47 (LoopF) and the reverse loop primer of SEQ ID NO: 48 (LoopB).
  • the carbapenemase-producing enterococcus is Escherichia coli , Klebsiella pneumonia , Pseudomonas spp. , Serratia spp ., Citro Citrobacter spp. , Acinetobacter spp. , It may be one or more selected from the group consisting of Morganella spp. , Providencia spp. And Enterobacter spp .
  • the present invention also provides a kit for detecting carbapenemase-producing intestinal bacteria comprising a primer set for loop-mediated isothermal amplification (LAMP).
  • LAMP loop-mediated isothermal amplification
  • the kit may further include a DNA polymerase for loop-mediated isothermal amplification reaction, dNTP (dATP, dCTP, dGTP and dTTP) mixture, buffer solution, and distilled water.
  • dNTP dATP, dCTP, dGTP and dTTP
  • the present invention also relates to the present invention.
  • the sample of step (a) may be obtained by feces or rectal swab.
  • the loop-mediated isothermal amplification reaction may be performed at 60 ° C to 70 ° C.
  • the present invention confirmed that the primer set for loop-mediated isothermal amplification reaction for the detection of carbapenemase-producing intestinal bacteria can be carried out within 1 hour with simple equipment, effectively detecting carbapenemase-producing intestinal bacteria. Since it was confirmed, it is possible to effectively provide information on whether or not to administer carbapenem.
  • LAMP loop-mediated isothermal amplification
  • FIG. 2 is data comparing the sensitivity of detecting carbapenemase-producing gut bacteria of two types of IMP gene loop-mediated isothermal amplification reactions with general PCR and real-time PCR.
  • 2A to 2C are results of detecting intestinal bacteria producing carbapenemase according to each sample sample.
  • IMP-1 refers to the primer set in Table 1
  • IMP-2 refers to the primer set in Table 2
  • the curve marked with ⁇ indicates a positive control
  • the curve marked with ⁇ indicates a negative control.
  • FIG. 3 is data comparing the sensitivity of detecting carbapenemase-producing gut bacteria of two types of VIM gene loop-mediated isothermal amplification reactions with general PCR and real-time PCR.
  • 3A to 3F are results of detecting intestinal bacteria producing carbapenemase according to each sample sample.
  • VIM-1 refers to the primer set in Table 3
  • VIM-2 refers to the primer set in Table 4
  • the curve marked with ⁇ indicates a positive control
  • the curve marked with X indicates a negative control.
  • FIG. 4 is data comparing the sensitivity of detecting carbapenemase-producing intestinal bacteria of two types of KPC gene loop-mediated isothermal amplification reactions with general PCR and real-time PCR.
  • 4A to 4E are results of detecting intestinal bacteria producing carbapenemase according to each sample sample.
  • KPC-1 refers to the primer set in Table 5
  • KPC-2 refers to the primer set in Table 6
  • the curve marked with o indicates a positive control
  • the curve marked with x indicates a negative control.
  • FIG. 5 is data comparing the sensitivity of detecting carbapenemase-producing intestinal bacteria of two types of NDM gene loop-mediated isothermal amplification reactions with general PCR and real-time PCR.
  • 5A to 5E are results of detecting intestinal bacteria producing carbapenem according to each sample sample.
  • NDM-1 refers to the primer set in Table 7
  • NDM-2 refers to the primer set in Table 8.
  • the curve marked with ⁇ indicates a positive control
  • the curve marked with X indicates a negative control.
  • Figure 6 is a result confirming the specificity of the detection of intestinal bacteria producing carbapenemase of the primer set for isothermal amplification reaction of the present invention. Curves marked with o indicate positive controls, curves marked with x indicate negative controls.
  • a primer set for a loop-mediated isothermal amplification reaction for quickly and effectively detecting intestinal bacteria producing carbapenemase was prepared.
  • the primer set showed sensitivity similar to that of a detection method using general PCR, and only 1 hour with simple equipment. It has the effect of efficiently detecting only intestinal bacteria producing carbapenemase within.
  • the present invention (1) SEQ ID NO: 1 to SEQ ID NO: primer set for detecting IMP gene consisting of the base sequence of 6;
  • KPC gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 29 to SEQ ID NO: 34;
  • NDM gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 43 to SEQ ID NO: 48; Provided is a primer set for loop-mediated isothermal amplification (LAMP) for detecting carbapenemase producing enterobacteriaceae (CPE), which is composed of any one or more primer sets.
  • LAMP loop-mediated isothermal amplification
  • CPE carbapenemase producing enterobacteriaceae
  • the "primer” of the present invention is a nucleic acid sequence having a short free 3'-hydroxyl group, which can form a complementary template and a base pair, and is a starting point for copying a template.
  • Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures and four different nucleoside triphosphates. PCR conditions, sense and antisense primer lengths can be modified based on those known in the art. These oligonucleotide primers can be easily prepared and used by those skilled in the art according to methods known in the art.
  • the "primer" of the present invention is a single-stranded oligonucleotide, suitable conditions (the presence of four different nucleoside triphosphates and polymerases such as DNA or RNA polymerase), suitable temperature and suitable Means acting as a starting point to initiate template-directed DNA synthesis under buffer.
  • Oligonucleotides used as primers can also include nucleotide analogs, such as phosphorothioates, alkylphosphorothioates or peptide nucleicacids or intercalating agents. It may include.
  • the suitable length of the primer is determined by the properties of the primer to be used, but it can be used in a length of 8 to 500 bp.
  • the primer need not be exactly complementary to the sequence of the template, but a complementary enough to form a hybridcomplex with the template can be used.
  • LAMP loop-mediated isothermal amplification
  • the primer set for IMP gene detection is a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, a forward inner primer of SEQ ID NO: 3 (FIP), a reverse internal primer of SEQ ID NO: 4 Back inner pimer (BIP), consisting of a forward loop primer (LoopF) of SEQ ID NO: 5 and a reverse loop primer (LoopB) of SEQ ID NO: 6,
  • the primer set for VIM gene detection includes a forward primer of SEQ ID NO: 15, a reverse primer of SEQ ID NO: 16, a forward inner primer of SEQ ID NO: 17 (FIP), and a reverse inner primer of SEQ ID NO: 18 (Back inner pimer; BIP) ), A forward loop primer of SEQ ID NO: 19 (LoopF) and a reverse loop primer of SEQ ID NO: 20 (LoopB),
  • the KPC gene detection primer set includes a forward primer of SEQ ID NO: 29, a reverse primer of SEQ ID NO: 30, a forward inner primer of SEQ ID NO: 31 (FIP), and a reverse inner primer of SEQ ID NO: 32 (Back inner pimer; BIP) ), A forward loop primer of SEQ ID NO: 33 (LoopF) and a reverse loop primer of SEQ ID NO: 34 (LoopB),
  • the NDM gene detection primer set includes a forward primer of SEQ ID NO: 43, a reverse primer of SEQ ID NO: 44, a forward inner primer of SEQ ID NO: 45 (FIP), and a reverse inner primer of SEQ ID NO: 46 (Back inner pimer; BIP). ), A forward loop primer of SEQ ID NO: 47 (LoopF) and a reverse loop primer of SEQ ID NO: 48 (LoopB).
  • the carbapenemase-producing enterococcal bacteria are Escherichia coli , Klebsiella pneumonia , Pseudomonas spp. , Serratia spp ., Citrobacter spp . ), Acinetobacter spp. It may be one or more selected from the group consisting of Morganella spp. , Providencia spp. , And Enterobacter spp.
  • Escherichia coli pneumococcal ( Klebsiella pneumonia ), Pseudomonas aeruginosa , Citrobacter freundii , Acinetobacter baumannii , Morganella morganii , Serratia marcescens , Serratia marcescens Expression of one or more genes of Enterobacter cloacae , Enterobacter aerogenes and Providencia stuartii , or IMP, VIM, KPC and NDM genes that produce carbapenemase Intestinal bacteria known to be capable of being detected without limitation.
  • two sets of primers for loop-mediated isothermal amplification reactions for IMP, VIM, KPC, and NDM are prepared, and sensitivity and efficiency for detecting intestinal bacteria produced by carbapenemase are produced.
  • primer sets corresponding to Tables 1 to 8 were prepared.
  • the primer set for Table 9 and Table 10 was prepared to compare the sensitivity and efficiency of detection of intestinal bacteria, thereby generating carbapenemase. Comparative experiments were performed using DNA isolated from intestinal bacteria.
  • IMP loop-mediated isothermal amplification reaction primer set-1 VIM loop-mediated isothermal amplification reaction primer set-1, KPC loop-mediated isothermal amplification reaction primer set-1 and NDM loop-mediated isothermal amplification reaction It was confirmed that the primer set-1 was suitable for detecting intestinal bacteria producing carbapenemase.
  • primer set can actually specifically detect only carbapenemase-producing intestinal bacteria
  • various types of general strains carbapenem-resistant intestinal bacteria ( Carbapenem resistant enterobacteriaceae (CRE), ESBL (Extended-spectrum beta-lactamases) and non-ESBL (non Extended-spectrum beta-lactamases) using isothermal amplification reaction in the same manner as in Example 2-2
  • CRE Carbapenem resistant enterobacteriaceae
  • ESBL Extended-spectrum beta-lactamases
  • non-ESBL non Extended-spectrum beta-lactamases
  • the present invention also provides a kit for detecting carbapenemase-producing intestinal bacteria comprising a primer set for loop-mediated isothermal amplification (LAMP).
  • LAMP loop-mediated isothermal amplification
  • the reagent for performing the amplification reaction is not particularly limited as long as it is usually included in the kit, but preferably, a DNA polymerase for loop-mediated isothermal amplification reaction, dNTP (dATP, dCTP, dGTP and dTTP) Mixtures, buffer solutions, and distilled water.
  • dNTP dATP, dCTP, dGTP and dTTP
  • the kit of the present invention may further include a user guide describing optimal conditions for performing the reaction.
  • the handbook is a printout that describes how to use the kit, for example, how to prepare a buffer, and the reaction conditions presented.
  • the guide may include a brochure or leaflet, a label attached to the kit, and a description on the surface of the package containing the kit.
  • the handbook may include information disclosed or provided through an electric medium such as the Internet.
  • the present invention also relates to the present invention.
  • Primer set for IMP gene detection consisting of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 6;
  • KPC gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 29 to SEQ ID NO: 34;
  • NDM gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 43 to SEQ ID NO: 48; Amplifying a target sequence by performing a loop-mediated isothermal reaction using a primer set for any one or more of the loop-mediated isothermal reactions;
  • the step (a) is a step of separating DNA from a sample, wherein the sample is obtained by a fecal or rectal swab collected from an organism, and the sample is homogenized and extracted from the biological sample as necessary. It may be a sample obtained by performing any necessary pretreatment. Such pretreatment can be selected by a person skilled in the art depending on the biological sample to be targeted.
  • DNA obtained from the sample can be obtained using methods well known in the art, such as using a commercially available DNA extraction kit from a sample obtained from an individual using feces or rectal swab. have.
  • an operation of amplifying the nucleotide can be performed as necessary.
  • the amplification operation can be performed, for example, through a PCR method using an enzyme such as DNA polymerase.
  • the step (b) is a step of performing a loop-mediated isothermal amplification reaction using DNA extracted from a sample as a template, using a primer set for loop-mediated isothermal amplification for detecting intestinal bacteria producing carbapenemase of the present invention. It is characterized in that it is carried out, preferably at 60 °C to 70 °C, more preferably at 65 °C.
  • the loop-mediated isothermal amplification reaction is similar to the conventional polymerase chain reaction (PCR) method, but the existing polymerase chain reaction repeatedly performs three steps of denaturation, conjugation, and elongation, thereby amplifying the target gene. While it is necessary to continuously change the temperature during the reaction process, the loop-mediated isothermal amplification reaction can be conjugated and stretched at a constant temperature. Since the loop-mediated isothermal amplification reaction uses Bst DNA polymerase having nucleic acid terminal hydrolase activity instead of Taq DNA polymerase, it is possible to induce denaturation of DNA's double helix structure without being dependent on heat.
  • PCR polymerase chain reaction
  • step (c) is a step of detecting amplification products, and each of the IMP, VIM, KPC and NDM genes is specifically amplified by the loop-mediated isothermal amplification primer, and IMP, VIM, KPC and When any of the NDM genes is amplified, it can be determined that it is an intestinal bacterium producing carbapenemase.
  • Whether or not amplification products are generated can be detected by labeling a detectable labeling material on the amplification target sequence, and as one specific example, the labeling material may be a material emitting fluorescence, phosphorescence, or radiation, but is not limited thereto.
  • the labeling material can be identified by the naked eye, a sensor or other means, preferably the labeling material is biotin, digoxigenin, dinitrophenol, aminoacetylfluorene , Sulfonate, FITC (Fluorescein isothiocyanate), rhodamine, aminomethylcoumarin, FAM (Carboxyl fluorescein-aminohexyl-amidite), TAMRA (Tetramethyl-6-Carboxyrhodamine) and cyanine It may be selected from one or more, but if it can function as a cover, the type is not particularly limited.
  • the present invention provides a method for providing information on whether or not to administer carbapenem using the method for detecting intestinal bacteria producing carbapenemase.
  • the primer set for the loop-mediated isothermal amplification reaction of the present invention if any of the IMP, VIM, KPC, and NDM genes is amplified, it can be determined that it is an intestinal bacterium producing carbapenemase, and in this case, carbapenem administration You can provide information to decide not to.
  • Example 1 Preparation of a primer set for loop-mediated isothermal amplification reaction for detection of intestinal bacteria producing carbapenemase
  • a primer set for loop-mediated isothermal amplification reactions for IMP, VIM, KPC, and NDM known as carbapenemase generating genes was prepared.
  • the loop-mediated isothermal amplification (LAMP) method is a method using a Bst DNA polymerase having a strand displacement DNA synthesis function, and loops using two pairs of specific primers. It is a method of continuously amplifying after forming.
  • forward primer, backward primer, forward inner pimer (FIP), reverse inner primer A primer set consisting of a back inner pimer (BIP), a forward loop primer (LoopF) and a reverse loop primer (LoopB) was prepared.
  • a primer set for loop-mediated isothermal amplification reactions for IMP, VIM, KPC, and NDM was prepared in each of two sets to select a primer set with high sensitivity and efficiency for detection of intestinal bacteria producing carbapenemase.
  • Primers were prepared by requesting Macrogen, and each primer sequence is shown in Tables 1 to 8 below.
  • the loop-mediated isothermal amplification reaction primer set prepared in Example 1 a general PCR primer set, and a real-time PCR primer set, it was intended to detect intestinal bacteria producing carbapenemase.
  • Samples were obtained using a rectal swab, and inoculated with the carbapenemase-producing enteric bacteria isolated from the samples were inoculated into MacConkey agar and cultured at 37 ° C. for over night.
  • the cultured strain was adjusted to 1.5 x 10 8 cells (MaF 0.5) using a turbidimeter, transferred to a 1.5 ml tube, boiled at 100 ° C for 10 minutes, and centrifuged at 13,000 rpm for 5 minutes.
  • the obtained DNA for 1.5 x 10 8 cells was prepared by serial dilution to 1.5 x 10. As shown in Table 11, intestinal bacteria expressing IMP, VIM, KPC and NDM genes in the sample were isolated, and a positive control for each of the carbapenemase-generating genes was used.
  • Example 2-1 using the DNA isolated in Example 2-1 as a template, a carbapenemase intestine is produced using the primer set for the loop-mediated isothermal amplification reaction prepared in Example 1, a general PCR primer set, and a real-time PCR primer set. Bacterial detection sensitivity was confirmed.
  • Bionic premix PCR tube (Bioneer premix PCR tube; BIONEER AccuPower PCR PreMix, K-2016-C), and use the primer set for each gene in Table 9 according to the product manual. Amplified.
  • 1 ⁇ l of Reactive Buffer (with 1.5 mM MgCl2) and 250 ⁇ M of dNTP each and 1 U of DNA polymerase ( Top DNApolymerase) are freeze-dried.
  • a PCR reaction solution of 20 ⁇ l consisting of 16 ⁇ l and 2 ⁇ l of Example 2-1 DNA was prepared, after 5 minutes and 1 cycle reaction at 94 ° C. for 30 seconds at 94 ° C., 56 ° C.
  • PCR was performed at 35 cycles for the IMP and VIM genes and 30 cycles for the KPC and NDM genes, and then each amplified product was amplified by electrophoresis (120 V / 35 min) using 1.5% agarose gel. .
  • the loop-mediated isothermal amplification reaction was performed according to the product manual using the NEB LAMP kit (WarmStart LAMP kit; E1700L), and analysis was performed using a real-time PCR analysis device (Bio-rad CFX connect Real-time system; 788BR06588).
  • DNA was amplified using a primer set for each gene in Tables 1 to 8, and a reaction solution was prepared as shown in Table 12, and amplification reaction was performed at 65 ° C for 60 minutes.
  • the detection sensitivity shows a large difference according to the primer sequence
  • the primer sets corresponding to Table 1, Table 3, Table 5, and Table 7 having high detection sensitivity and general PCR and real-time PCR analysis sensitivity are shown below. It was summarized as Table 13.
  • IMP loop-mediated isothermal amplification reaction primer set-1 VIM loop-mediated isothermal amplification reaction primer set-1, KPC loop-mediated isothermal amplification reaction primer set-1 and NDM loop-mediated isothermal amplification reaction It was confirmed that the primer set-1 was suitable for detecting intestinal bacteria producing carbapenemase.
  • carbapenem resistant intestinal bacteria in order to confirm that the primer set for loop-mediated isothermal amplification reaction which confirmed the effect in Example 2 can actually detect only carbapenemase-producing intestinal bacteria
  • a loop-mediated isothermal amplification reaction was performed in the same manner as in Example 2-2 using enterobacteriaceae (CRE), ESBL (Extended-spectrum beta-lactamases) and non-ESBL (non Extended-spectrum beta-lactamases).
  • CRE enterobacteriaceae
  • ESBL Extended-spectrum beta-lactamases
  • non-ESBL non Extended-spectrum beta-lactamases
  • loop-mediated isothermal amplification primer set of the present invention can selectively detect only intestinal bacteria producing carbapenemase.
  • the present invention confirmed that the primer set for loop-mediated isothermal amplification reaction for the detection of carbapenemase-producing intestinal bacteria can be carried out within 1 hour with simple equipment, effectively detecting carbapenemase-producing intestinal bacteria. Since it was confirmed, it can be usefully used as a diagnostic kit for detecting intestinal bacteria producing carbapenemase.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Ensemble d'amorces pour une réaction d'amplification isothermique médiée par boucle pour diagnostiquer des entérobactéries productrices de carbapénèmase, et utilisation correspondante. Plus particulièrement, la présente invention porte sur un ensemble d'amorces pour une réaction d'amplification isothermique médiée par boucle pour IMP, VIM, KPC, et NDM, qui sont connus pour être des gènes produisant des carbapénèmases; et un procédé de diagnostic d'entérobactéries productrices de carbapénémases correspondant. Selon la présente invention, il a été découvert qu'un ensemble d'amorces pour une réaction d'amplification isothermique médiée par boucle pour détecter des entérobactéries productrices de carbapénèmases peut être utilisé pour effectuer un test en une heure à l'aide uniquement d'un équipement simple, et détecter efficacement uniquement les entérobactéries productrices de carbapénèmases, et peut ainsi fournir efficacement des informations sur l'opportunité d'administrer un carbapénème.
PCT/KR2019/013707 2018-11-16 2019-10-18 Ensemble d'amorces pour une réaction d'amplification isothermique médiée par boucle pour diagnostiquer des entérobactéries productrices de carbapénèmase, et utilisation correspondante WO2020101194A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020180141717A KR102110408B1 (ko) 2018-11-16 2018-11-16 카바페넴아제 생성 장내세균 진단을 위한 루프 매개 등온증폭 반응용 프라이머 세트 및 이의 용도
KR10-2018-0141717 2018-11-16

Publications (1)

Publication Number Publication Date
WO2020101194A1 true WO2020101194A1 (fr) 2020-05-22

Family

ID=70730127

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2019/013707 WO2020101194A1 (fr) 2018-11-16 2019-10-18 Ensemble d'amorces pour une réaction d'amplification isothermique médiée par boucle pour diagnostiquer des entérobactéries productrices de carbapénèmase, et utilisation correspondante

Country Status (2)

Country Link
KR (1) KR102110408B1 (fr)
WO (1) WO2020101194A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112458153A (zh) * 2020-12-01 2021-03-09 重庆医科大学附属第一医院 一种检测肠杆菌目五大类碳青霉烯酶基因及其亚型的lamp引物及其试剂盒

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024005458A1 (fr) * 2022-06-27 2024-01-04 주식회사 씨젠 Procédé d'amplification d'acide nucléique cible à l'aide d'acide bêta-hydroxy

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130005208A (ko) * 2011-07-05 2013-01-15 (주)지노첵 리얼타임 ｐｃｒ을 이용한 카바페넴 내성 장내균 검사 방법 및 키트
CN103614465A (zh) * 2013-11-06 2014-03-05 南方医科大学 用于检测革兰阴性杆菌常见碳青霉烯酶基因的lamp引物、试剂盒及其检测方法
WO2014178401A1 (fr) * 2013-05-01 2014-11-06 学校法人帝京大学 PROCÉDÉ DE DÉTECTION DE BACTÉRIE RÉSISTANTE AUX MÉDICAMENTS À BASE DE CARBAPENÈME (BACTÉRIE PRODUISANT DES β-LACTAMASES DE TYPE KPC)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130005208A (ko) * 2011-07-05 2013-01-15 (주)지노첵 리얼타임 ｐｃｒ을 이용한 카바페넴 내성 장내균 검사 방법 및 키트
WO2014178401A1 (fr) * 2013-05-01 2014-11-06 学校法人帝京大学 PROCÉDÉ DE DÉTECTION DE BACTÉRIE RÉSISTANTE AUX MÉDICAMENTS À BASE DE CARBAPENÈME (BACTÉRIE PRODUISANT DES β-LACTAMASES DE TYPE KPC)
CN103614465A (zh) * 2013-11-06 2014-03-05 南方医科大学 用于检测革兰阴性杆菌常见碳青霉烯酶基因的lamp引物、试剂盒及其检测方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DOYLE, D. ET AL.: "L aboratory Detection of Enterobacteriaceae That Produce Carbapenemases", J. CLIN. MICROBIOL, vol. 50, no. 12, December 2012 (2012-12-01), pages 3877 - 3880, XP002710011, DOI: 10.1128/JCM.02117-12 *
NOTOMI, T. ET AL.: "Loop-mediated isothermal amplification of DNA", NUCLEIC ACIDS RES., vol. 28, no. 12, 15 June 2000 (2000-06-15), pages E63, XP003018858, DOI: 10.1093/nar/28.12.e63 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112458153A (zh) * 2020-12-01 2021-03-09 重庆医科大学附属第一医院 一种检测肠杆菌目五大类碳青霉烯酶基因及其亚型的lamp引物及其试剂盒

Also Published As

Publication number Publication date
KR102110408B1 (ko) 2020-05-13

Similar Documents

Publication Publication Date Title
JP5986043B2 (ja) クラミジア・トラコマチスを検出するためのプライマー及びプローブ配列
WO2020101194A1 (fr) Ensemble d'amorces pour une réaction d'amplification isothermique médiée par boucle pour diagnostiquer des entérobactéries productrices de carbapénèmase, et utilisation correspondante
WO2011025262A2 (fr) Composition pour détecter séparément le complexe mycobacterium tuberculosis et le genre mycobacterium, et procédé pour détecter simultanément mycobacterium tuberculosis et le genre mycobacterium par réaction de polymérase en chaîne multiplexée en temps réel utilisant celle-ci
WO2017099445A1 (fr) Marqueur génétique permettant de distinguer et de détecter des bactéries à l'origine de l'infection à edwardsiella et de la streptococcocie chez le poisson, et procédé de différenciation et de détection de telles bactéries utilisant ledit marqueur
WO2017122897A1 (fr) Marqueur génétique servant à détecter le virus responsable de l'iridovirose de la daurade japonaise, et procédé de détection du virus causal utilisant le marqueur
WO2018074762A1 (fr) Procédé et kit de diagnostic sur la base d'une plate-forme de test quantamatrix, capables de détecter et d'identifier des bactéries à gram positif et à gram négatif et des espèces de candida et de déterminer la résistance aux antibiotiques simultanément
US20160060684A1 (en) Rapid salmonella serotyping assay
CN105950778A (zh) 依赖mert-lamp技术检测粪肠球菌和金黄色葡萄球菌的核苷酸序列
WO2021177773A2 (fr) Composition pour le diagnostic du sars-cov-2, kit, et procédé pour le diagnostic du sars-cov-2 l'utilisant
EP1651773A1 (fr) Diagnostic d'escherichia coli (dec) et de shigella spp diarrheogenes
WO2017039144A1 (fr) Trousse et procédé de détection de bactéries entraînant des maladies sexuellement transmises
WO2021162228A1 (fr) Ensemble d'amorces pour l'analyse de la résistance aux médicaments du vih-1, kit le comprenant et procédé d'analyse l'utilisant
JP5900979B2 (ja) 細胞膨張化致死毒を標的としたカンピロバクター属細菌の検出
WO2024080483A1 (fr) Composition fondée sur la technique crispr-cas pour la détection de la listeria monocytogenes et procédé de détection de la listeria monocytogenes l'utilisant
WO2022145658A1 (fr) Composition pour diagnostiquer une maladie de tsutsugamushi et kit la comprenant
WO2011162441A1 (fr) Paires d'amorces pour détecter orientia tsutsugamushi et procédé de détection utilisant celles-ci
US9115408B2 (en) Rapid Salmonella serotyping assay
WO2022211238A1 (fr) Procédé de diagnostic moléculaire basé sur l'arn 16s pour la tuberculose pour le suivi de l'efficacité du traitement, et son application
WO2012002598A1 (fr) Amorce et sonde pour le diagnostic de la tuberculose, trousse comprenant celles-ci et méthode de diagnostic de la tuberculose à l'aide de la trousse
KR102388060B1 (ko) 결핵균 및 베이징 계통 결핵균의 감별을 위한 조성물 및 이를 이용한 결핵균 검출 방법
WO2015037769A1 (fr) Composition, méthode et kit pour diagnostiquer une septicémie
WO2021075912A1 (fr) Ensemble d'amorces pour une réaction d'amplification multi-isotherme à haute sensibilité capable de cribler et de détecter simultanément mycobacterium tuberculosis et des mycobactéries non tuberculeuses
WO2012134063A2 (fr) Amorce pour la détection de bactéries pathogènes de maladies infectieuses bactériennes orales, et utilisation de celle-ci
WO2021141179A1 (fr) Oligonucléotide et plasmide pour la détection simultanée de quatre sérotypes du virus de la dengue, et procédé d'analyse du sérotype du virus de la dengue utilisant celui-ci
EP2231866A1 (fr) Procédés et oligonucléotides pour la détection de bactéries provoquant une mastite

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19885674

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19885674

Country of ref document: EP

Kind code of ref document: A1