WO2026005351A1 - Procédé de détection d'acide nucléique d'un micro-organisme cible à partir d'un échantillon cutané à l'aide de corynebacterium spp. ou cutibacterium spp. comme témoin interne endogène - Google Patents

Procédé de détection d'acide nucléique d'un micro-organisme cible à partir d'un échantillon cutané à l'aide de corynebacterium spp. ou cutibacterium spp. comme témoin interne endogène

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Publication number
WO2026005351A1
WO2026005351A1 PCT/KR2025/008138 KR2025008138W WO2026005351A1 WO 2026005351 A1 WO2026005351 A1 WO 2026005351A1 KR 2025008138 W KR2025008138 W KR 2025008138W WO 2026005351 A1 WO2026005351 A1 WO 2026005351A1
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nucleic acid
spp
corynebacterium
sample
cutibacterium
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English (en)
Korean (ko)
Inventor
이현지
김현중
이혜민
윤소라
이연지
이도경
편린아
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Seegene Inc
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Seegene Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—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
    • C12Q1/6851—Quantitative amplification
    • 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
    • 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

Definitions

  • the present invention relates to a method for detecting a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid, and to a composition for nucleic acid amplification used in the method.
  • Human diseases can be detected through in vitro and in vitro diagnostics.
  • In vitro diagnostics analyze the cause of disease through X-rays, CT scans, and other methods, while in vitro diagnostics analyze the cause of disease through urine, blood, tissue cells, and other methods.
  • In vitro diagnostics include immunochemical diagnostics, self-blood glucose monitoring, point-of-care diagnostics, and molecular diagnostics.
  • molecular diagnostics directly examines genes using techniques like polymerase chain reaction (PCR). This involves extracting nucleic acids containing the pathogen's genetic information from samples such as saliva, blood, or stool from infected individuals, then amplifying these nucleic acids to confirm the presence of disease.
  • PCR polymerase chain reaction
  • molecular diagnostics In molecular diagnostics, accurate and appropriate sample collection is essential for accurate test results. Insufficient sample volume or contamination during sample collection can lead to inaccurate test results, increasing retest rates and delaying test result reporting. Furthermore, as described above, molecular diagnostics involves extracting and amplifying nucleic acids from a sample. If loss occurs during the nucleic acid extraction process or if the sample solution contains substances that inhibit the amplification reaction (e.g., heparin, detergents, protein denaturants, or organic solvents), amplification efficiency will be reduced, preventing sufficient amplification of pathogen nucleic acids. In such cases, despite the presence of pathogens in the sample, the inability to amplify pathogen nucleic acids can result in false negative results. Furthermore, even when using the same sample, different results can be obtained depending on the degree of loss during the nucleic acid extraction process.
  • substances that inhibit the amplification reaction e.g., heparin, detergents, protein denaturants, or organic solvents
  • the present inventors have diligently studied and endeavored to develop a novel internal control that can improve the accuracy of detection by minimizing false negative and false positive judgments in a method for detecting target microorganisms from samples collected from the skin using nucleic acid amplification.
  • the present inventors have experimentally demonstrated that the nucleic acid of Corynebacterium spp. or Cutibacterium spp. can be successfully used as an internal control for the sample collection process, the nucleic acid extraction process from the sample, and/or the nucleic acid amplification process of the extracted nucleic acid in a method for detecting target microorganisms from samples collected from the skin using nucleic acid amplification, thereby completing the present invention.
  • the present invention provides a method for detecting a nucleic acid of a target microorganism from a sample collected from the skin using a bacterial nucleic acid selected from the normal skin flora as an endogenous internal control nucleic acid, comprising the following steps:
  • the present inventors have conducted extensive research efforts to develop a novel internal control that can be used in a method for detecting target microorganisms from skin samples using nucleic acid amplification.
  • a novel protocol for detecting target microorganism nucleic acids from skin samples was established using bacterial nucleic acids selected from the normal skin flora as internal control nucleic acids.
  • bacterial nucleic acids selected from the normal skin flora can be used as internal control nucleic acids for the sample collection process, the nucleic acid extraction process, and/or the nucleic acid amplification process of the extracted nucleic acids.
  • normal skin flora as used herein means the entire community of bacteria formed on the skin of a human or animal.
  • the normal skin flora is the normal skin flora of human skin.
  • the normal skin flora may include, but is not limited to, Corynebacterium , Cutibacterium , Staphylococcus , Enhydrobacter , and Micrococcus . More specifically, the normal flora of the skin includes Corynebacterium spp., Cutibacterium spp., Staphylococcus spp., Enhydrobacter spp., and Micrococcus spp., and more specifically, Corynebacterium tuberculostearicum , Corynebacterium simulans , Corynebacterium afermentans , Corynebacterium fastidiosum , Corynebacterium resistens , Cutibacterium acnes , Staphylococcus epidermidis , Staphylococcus capitis , Staphylococcus hominis , Staphylococcus haemolyticus , Staphylococcus warneri ,
  • the present invention provides a method for detecting a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid, comprising the following steps:
  • the method of the present invention includes the step of (a) collecting and preparing a sample from the skin.
  • sample in this specification means a sample obtained from a human or animal subject for which nucleic acid of a target microorganism is to be detected from a sample collected from the skin by a nucleic acid amplification method.
  • the sample is a sample obtained from a test subject of a human or animal suspected of being infected with a pathogenic microorganism.
  • the animals include, but are not limited to, primates, livestock (e.g., pigs, sheep, cows, horses, and donkeys), laboratory animals (e.g., rats, mice, guinea pigs, hamsters, and rabbits), pet animals (e.g., dogs and cats), farmed wild animals (e.g., squirrels, foxes, kangaroos, and deer), and birds.
  • livestock e.g., pigs, sheep, cows, horses, and donkeys
  • laboratory animals e.g., rats, mice, guinea pigs, hamsters, and rabbits
  • pet animals e.g., dogs and cats
  • farmed wild animals e.g., squirrels, foxes, kangaroos, and deer
  • the preparation of the sample further comprises a step of extracting nucleic acid from the sample.
  • nucleic acid or “nucleic acid molecule” as used herein refers to a deoxyribonucleotide or ribonucleotide polymer in single-stranded or double-stranded form, wherein the nucleotides include derivatives of natural nucleotides, non-natural nucleotides, or modified nucleotides that can function in the same manner as naturally occurring nucleotides.
  • Nucleic acids can be extracted from the above samples using various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001).
  • various nucleic acid extraction kits are commercially available depending on the type of sample, and those skilled in the art can extract nucleic acids from various samples using commercially available kits.
  • the method of the present invention includes a step of performing an amplification reaction of the nucleic acid in the sample using (i) a pair of primers for amplifying the nucleic acid of the target microorganism; and (ii) a pair of primers for amplifying the nucleic acid of Corynebacterium spp. or Cutibacterium spp., which is an endogenous internal control nucleic acid.
  • the amplification reaction of the nucleic acid may additionally include (i) a probe for detecting the nucleic acid of the target microorganism; and (ii) a probe for detecting the nucleic acid of Corynebacterium spp. or Cutibacterium spp.
  • primer as used herein means an oligonucleotide that acts as an initiation point for synthesis under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid chain (template), i.e., the presence of nucleotides and a polymerizing agent such as a nucleic acid polymerase, and conditions of suitable temperature and pH.
  • template i.e., the presence of nucleotides and a polymerizing agent such as a nucleic acid polymerase, and conditions of suitable temperature and pH.
  • probe as used herein means a single-stranded nucleic acid molecule comprising a portion or portions substantially complementary to a target nucleic acid sequence.
  • the primers or probes used in the present invention may include natural NMPs (i.e., AMP, GMP, CMP, and UMP), natural dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides.
  • natural NMPs i.e., AMP, GMP, CMP, and UMP
  • natural dNMPs i.e., dAMP, dGMP, dCMP, and dTMP
  • modified nucleotides i.e., dAMP, dGMP, dCMP, and dTMP
  • the primer must be sufficiently long to prime the synthesis of the extension product in the presence of the polymerizing agent.
  • the appropriate primer length depends on several factors, such as temperature, application, and primer source.
  • annealing or “priming” herein refers to the juxtaposition of an oligonucleotide or nucleic acid to a template nucleic acid, which juxtaposition causes a polymerase to polymerize the nucleotides to form a nucleic acid molecule complementary to the template nucleic acid or a portion thereof.
  • complementary means sufficiently complementary that a primer or probe selectively hybridizes to a target nucleic acid molecule under given annealing or hybridization conditions, and includes both substantially complementary and perfectly complementary, and specifically means perfectly complementary.
  • target nucleic acid refers to a nucleic acid molecule that is ultimately to be amplified or detected, and is annealed or hybridized with a primer under specific hybridization conditions.
  • target microorganism refers to a microorganism to be detected in a sample collected from the skin.
  • nucleic acid of target microorganism means the nucleic acid of a microorganism to be detected in a sample collected from the skin.
  • the target microorganism to be detected through the method of the present invention refers to a microorganism present in a sample collected from the skin of a human or animal, and the microorganism may include, but is not limited to, bacteria, yeast, fungi, viruses, protozoans, etc.
  • the target microorganism is a drug-resistant microorganism, a dermatophyte, or methicillin-resistant staphylococcus aureus (MRSA).
  • MRSA methicillin-resistant staphylococcus aureus
  • the drug-resistant microorganism is a multidrug resistant organism, and more specifically includes Acinetobacter baumannii , Pseudomonas aeruginosa , Candida auris , and Stenotrophomonas maltophilia .
  • the nucleic acid of the target microorganism may include, but is not limited to, a DNA molecule or an RNA molecule.
  • the nucleic acid of the target microorganism may include a resistance gene that confers drug resistance, and may include, for example, a mutation of a gene, a resistance gene mediated by a plasmid or a transposon.
  • the nucleic acid of Corynebacterium spp. or Cutibacterium spp. used as the internal control nucleic acid is different from the nucleic acid of the target microorganism to be detected according to the method of the present invention.
  • the method for detecting nucleic acids of target microorganisms can detect 1 to 30, specifically 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 target microorganism nucleic acids simultaneously, and more specifically, can detect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 target microorganism nucleic acids, but is not limited thereto.
  • the nucleic acid amplification reaction may use 1 to 30 pairs, specifically 1 to 25 pairs, 1 to 20 pairs, 1 to 15 pairs, 1 to 10 pairs or 1 to 5 pairs of primer pairs for amplification of target microbial nucleic acids, and more specifically, 1 pair, 2 pairs, 3 pairs, 4 pairs, 5 pairs, 6 pairs, 7 pairs, 8 pairs, 9 pairs, 10 pairs, 15 pairs, 20 pairs, 25 pairs or 30 pairs of primer pairs for amplification of target microbial nucleic acids, but is not limited thereto.
  • hybridization refers to the formation of a double-stranded polynucleotide through non-covalent bonding between complementary nucleotide sequences of two single-stranded polynucleotides under certain hybridization conditions or stringent conditions.
  • Hybridization can occur when two nucleic acid sequences have perfect complementarity at the site of hybridization (the site where the double-stranded DNA is formed) or when there is a mismatch (e.g., a mismatch of 1 to 4 bases).
  • the degree of complementarity required for hybridization can vary depending on the conditions of the hybridization reaction, particularly temperature.
  • hybridization and “annealing” are not different and are used interchangeably in this specification.
  • Amplification of target nucleic acid molecules can be performed using various primer-assisted nucleic acid amplification methods known in the art. Specifically, target nucleic acid amplification is performed using polymerase chain reaction (PCR), a PCR method disclosed in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159. Other examples include ligase chain reaction (LCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), strand displacement amplification (SDA) (Walker, et al. Nucleic Acids Res.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • DNA polymerases can be used for the nucleic acid amplification of the present invention, including E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase.
  • the DNA polymerase is a thermostable DNA polymerase obtainable from various bacterial species, including Thermus aquaticus ( Taq ), Thermus thermophilus , Thermus filiformis , Thermus flavus, Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus , Thermus igniterrae , Thermus lacteus , Thermus oshimai, Thermus ruber, Thermus rubens , Thermus scotoductus , Thermus silvanus , Thermus species Z05, and Thermus species sps 17.
  • amplification of the nucleic acid can be performed by performing PCR.
  • the amplification reaction can be performed by a fast PCR method.
  • fast PCR refers to a PCR method that increases the speed of PCR compared to a general PCR method.
  • the fast PCR can be achieved by controlling various factors such as the extension rate of the DNA polymerase, the temperature change rate (ramp speed) of the thermal cycler, and the complexity of the template.
  • the extension rate of the DNA polymerase used in general PCR, which has a standard extension rate of 1 kb per minute
  • a Taq DNA polymerase with a standard extension rate of 2-4 kb per minute can be used to achieve fast PCR.
  • the nucleic acid of Corynebacterium spp. and Cutibacterium spp. may include a nucleotide sequence encoding 16s rRNA or gyrA (DNA gyrase subunit A).
  • the nucleic acid of the Corynebacterium spp. may include a nucleotide sequence encoding 16s rRNA, or the nucleic acid of the Cutibacterium spp. may include a nucleotide sequence encoding gyrA (DNA gyrase subunit A).
  • the nucleic acid of the Corynebacterium spp. comprises the sequence 1 in the sequence listing, or a complementary sequence thereof, or the nucleic acid of the Cutibacterium spp. comprises the sequence 6 in the sequence listing, or a complementary sequence thereof.
  • the Corynebacterium spp. is Corynebacterium tuberculostearicum , Corynebacterium simulans , Corynebacterium afermentans , Corynebacterium fastidiosum , or Corynebacterium resistens , or the Cutibacterium spp. is Cutibacterium acnes , and more specifically, the Corynebacterium spp. is Corynebacterium tuberculostearicum , or the Cutibacterium spp. is Cutibacterium acnes .
  • the 16s rRNA gene sequence of Corynebacterium tuberculostearicum can be found in GenBank accession number X84247.1, and that of Cutibacterium acnes
  • the gyrA gene sequence can be found in GenBank accession number KX650495.1.
  • the nucleic acid of Corynebacterium spp. or Cutibacterium spp. can be used as an internal control for (i) the step of collecting a sample, (ii) the step of extracting nucleic acid from the sample, and/or (iii) the step of amplifying the extracted nucleic acid.
  • the present inventors devised a method for detecting the nucleic acid of a target microorganism in a sample collected from the skin using the nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid.
  • the nucleic acid of Corynebacterium spp. or Cutibacterium spp. is amplified simultaneously with the amplification of the target microbial nucleic acid from a sample collected from the skin in the same reaction vessel.
  • the nucleic acid of Corynebacterium spp. or Cutibacterium spp. can be used as an internal control in each of the above steps. The role of the internal control in each step is described in detail as follows.
  • the sample is not collected properly during the sampling process, for example, if the amount of sample collected is too small to detect nucleic acids, the nucleic acids of the target microorganism may not be detected even if they are present in the sample collected from the skin. This may ultimately result in a false negative result, and such false negative results can be determined by confirming the presence or absence of nucleic acids of Corynebacterium spp. or Cutibacterium spp. as an internal control.
  • nucleic acid loss occurs during the nucleic acid extraction step, i.e., if the amount of nucleic acid extracted as a template for the amplification reaction is insufficient, the efficiency of the amplification reaction may be reduced, resulting in undetectable nucleic acid and false negative results.
  • the presence or absence of nucleic acid from Corynebacterium spp. or Cutibacterium spp. as an internal control can be used to determine whether a false negative result has occurred.
  • the amplification efficiency may be reduced despite the presence of the target nucleic acid, resulting in a false negative result without detecting the nucleic acid amplification.
  • the presence or absence of a false negative result can be determined by confirming the presence or absence of the nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an internal control.
  • the method according to the present invention further comprises a step of amplifying nucleic acid in the collected sample using a pair of primers for amplifying nucleic acid of Corynebacterium spp. or Cutibacterium spp., which is an endogenous internal control nucleic acid, thereby determining whether the collection of the sample is valid or invalid.
  • the method according to the present invention further comprises a step of amplifying nucleic acid extracted from the sample using a pair of primers for amplifying nucleic acid of Corynebacterium spp. or Cutibacterium spp., which is an endogenous internal control nucleic acid, thereby determining whether the extraction is valid or invalid.
  • the amplification reaction of a nucleic acid is carried out in the presence of a label or labeled oligonucleotide (labeled primer or labeled probe) capable of providing a signal dependent on the presence of the nucleic acid to be detected.
  • a label or labeled oligonucleotide labeled primer or labeled probe
  • a signal may be provided from a label during the process of amplifying a nucleic acid to be detected, or a signal may be provided after amplification is completed.
  • the method of the present invention includes the step of (c) detecting the result of the amplification reaction.
  • detection of the amplification reaction result can be performed by a post-amplification detection method or a real-time detection method.
  • the above post-amplification detection method is a method for detecting amplification products after nucleic acid amplification.
  • Post-amplification detection methods include, but are not limited to, methods for separating amplification products by size difference (e.g., electrophoresis) or by immobilizing the amplification products.
  • the above post-amplification detection method may use a post-PCR melting analysis (US Patent No. 5,871,908, US Patent No. 6,174,670 and WO 2012/096523) that monitors the fluorescence intensity while raising or lowering the temperature at a certain interval after amplification of the target nucleic acid sequence and then detects the amplification product by the melting profile.
  • a post-PCR melting analysis US Patent No. 5,871,908, US Patent No. 6,174,670 and WO 2012/096523
  • the above post-amplification detection method or real-time detection method may use a label or labeled oligonucleotide that provides a signal dependent on the presence of the nucleic acid to be detected.
  • detection of an amplified nucleic acid can be performed by detecting a signal provided from a label during the process of amplifying the nucleic acid to be detected or by detecting a signal provided after amplification of the nucleic acid to be detected is completed.
  • Examples of methods using labeled primers include the Sunrise primer method (Nazarenko et al, 2516-2521 Nucleic Acids Research, 1997, v.25 no.12, and U.S. Pat. No. 6,117,635), the Scorpion primer method (Whitcombe et al, 804-807, Nature Biotechnology v.17 AUGUST 1999, and U.S. Pat. No. 6,326,145), and the TSG primer method (WO 2011/078441).
  • dimer formed dependently on the presence of a target nucleic acid sequence can be obtained by various methods, for example, Invader assay (U.S. Patent Nos.
  • a method can be used to detect one or more target nucleic acid sequences using a single type of label by detecting signals at different temperatures. This is disclosed in WO 2015/147412, WO 2016/093619, and WO 2016/093620, all of which are incorporated herein by reference.
  • the amplification reaction may additionally include (i) a probe for detecting a nucleic acid of the target microorganism; and (ii) a probe for detecting a nucleic acid of the Corynebacterium spp. or Cutibacterium spp.
  • the amplification reaction may include 1 to 30, specifically 1 to 25, 1 to 20, 1 to 15, 1 to 10 or 1 to 5 target microorganism nucleic acid detection probes, and may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 target microorganism nucleic acid detection probes, but is not limited thereto.
  • the primer pair or probe of the nucleic acid of Corynebacterium spp. and Cutibacterium spp. comprises a nucleotide sequence that specifically hybridizes to a nucleotide sequence encoding 16s rRNA or gyrA (DNA gyrase subunit A), or a complementary sequence thereof.
  • the primer pair or probe of the nucleic acid of the Corynebacterium spp. comprises a nucleotide sequence that specifically hybridizes to a nucleotide sequence encoding 16s rRNA, or a complementary sequence thereof, or the primer pair or probe of the nucleic acid of the Cutibacterium spp. comprises a nucleotide sequence that specifically hybridizes to a nucleotide sequence encoding gyrA (DNA gyrase subunit A), or a complementary sequence thereof.
  • gyrA DNA gyrase subunit A
  • the term “specifically hybridizes” as used herein means that two or more molecules interact with each other through covalent or non-covalent bonds, for example, the binding of a single-stranded nucleotide molecule having a single-stranded target sequence and a nucleotide sequence complementary thereto.
  • the method of the present invention comprises the steps of (d) determining the validity of the amplification reaction of the target microorganism nucleic acid from the result of the amplification reaction of the internal control nucleic acid; and (i) determining whether the nucleic acid of the target microorganism is present in the sample based on the determined validity and (ii) the result of the amplification reaction of the target microorganism nucleic acid.
  • the validity of the amplification reaction result of the target microbial nucleic acid can be determined based on the amplification reaction result of the internal control group among the results of the amplification reaction.
  • the nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an internal control according to the present invention should always be amplified and detected by an amplification reaction, regardless of the presence or absence of the nucleic acid of the target microorganism.
  • nucleic acid of Corynebacterium spp. or Cutibacterium spp. when a nucleic acid of Corynebacterium spp. or Cutibacterium spp. is detected as an internal control, it can be determined that the sample collection, nucleic acid extraction, and/or nucleic acid amplification reaction were successfully performed, and the result of the amplification reaction of the target microorganism nucleic acid can be determined to be valid.
  • the nucleic acid of Corynebacterium spp. or Cutibacterium spp. is not detected as an internal control, it can be determined that a problem occurred in the process of collecting a sample, extracting nucleic acid, and/or amplifying nucleic acid, and the result of the amplification reaction of the target microorganism nucleic acid can be determined to be an invalid result, i.e., invalid.
  • invalid result refers to a result in which the detection result cannot be interpreted and is therefore treated as invalid, and the subject of a sample determined to have an invalid result can re-conduct the test for detection from the sample collection stage.
  • the result of the amplification reaction of the target microbial nucleic acid can be determined to be invalid.
  • detection of the target microorganism nucleic acid means the presence (positive) of the target microorganism nucleic acid
  • non-detection of the target microorganism nucleic acid in the amplification reaction result means the absence (negative) of the target microorganism nucleic acid
  • the result in which the target microbial nucleic acid is not detected can be judged to be invalid.
  • the result in which the target microbial nucleic acid is detected can be judged to be invalid.
  • nucleic acid amplification efficiency varies depending on the target nucleic acid, and amplification efficiency also varies within the same tube depending on various factors such as the initial amount of nucleic acid in the sample and the size of the amplified product.
  • nucleic acid of the target microorganism When the nucleic acid of the target microorganism is present in a relatively large amount compared to the nucleic acid of the internal control, during the amplification process, essential reagents (e.g., polymerase, dNTP, etc.) are consumed while amplifying the nucleic acid of the excess target microorganism, and the amplification efficiency of the nucleic acid of the internal control is relatively reduced, so that the amplification of the nucleic acid of the internal control may not be detected.
  • essential reagents e.g., polymerase, dNTP, etc.
  • the nucleic acid of the target microorganism may be amplified, while amplification of the endogenous internal control nucleic acid may not be detected.
  • the method of the present invention can be performed by adding an exogenous internal control to a sample collected after sample collection in addition to the internal control described above, and can be performed by including an external positive control and/or an external negative control in the nucleic acid amplification step.
  • the above-mentioned external internal control can be used as a control for the nucleic acid extraction process, the above-mentioned external positive control can be used as a control for the nucleic acid amplification process, and the above-mentioned external negative control can be used as a control for sample contamination and non-specific reactions.
  • the external internal control can be added to the sample after sample collection together with the internal control according to the present invention and used. If the external internal control is detected and the internal control according to the present invention is not detected, the sample collection process can be determined to have failed.
  • the present invention provides a composition for amplifying a nucleic acid of a target microorganism from a sample collected from the skin using a bacterial nucleic acid selected from the normal skin flora as an endogenous internal control nucleic acid, comprising:
  • a pair of primers for amplifying nucleic acids of bacteria selected from the normal skin flora.
  • composition for amplifying a nucleic acid of a target microorganism from a sample collected from the skin using a bacterial nucleic acid selected from the normal skin flora as an endogenous internal control nucleic acid is manufactured to carry out the above-described aspect, "a method for detecting a nucleic acid of a target microorganism from a sample collected from the skin using a bacterial nucleic acid selected from the normal skin flora as an endogenous internal control nucleic acid,” and therefore, the description of common contents between them is omitted to avoid excessive complexity of the present specification.
  • the present invention provides a composition for amplifying a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid, comprising:
  • composition for amplifying a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid which is another embodiment of the present invention, is manufactured to carry out the "method for detecting a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid", and therefore, the common contents between them are omitted in order to avoid excessive complexity of the present specification.
  • composition of the present invention described herein may optionally include reagents necessary for performing a target amplification reaction (e.g., a PCR reaction), such as a buffer, a DNA polymerase cofactor, and deoxyribonucleotide-5-triphosphate.
  • a target amplification reaction e.g., a PCR reaction
  • the composition of the present invention may also include various polynucleotide molecules, a reverse transcriptase, various buffers and reagents, and an antibody that inhibits DNA polymerase activity.
  • the composition may also include reagents necessary for performing positive and negative control reactions. The optimal amount of reagents to be used in a particular reaction can be readily determined by one of ordinary skill in the art having learned the disclosure herein.
  • the composition of the present invention is manufactured in a separate package or compartment containing the aforementioned components.
  • a nucleic acid of Corynebacterium spp. or Cutibacterium spp. is used as an endogenous internal control nucleic acid.
  • the internal control according to the present invention can be used as an internal control in a sample collection process, an internal control in a nucleic acid extraction process, and an internal control in a nucleic acid amplification process.
  • Figure 1 is a flowchart of processes for implementing the method of the present invention according to one embodiment of the present invention.
  • Example 1 From a sample taken from the skin Corynebacterium tuberculostearicum Check the detection rate
  • the present inventors confirmed that the nucleic acid of Corynebacterium tuberculostearicum , a bacterium selected from normal skin flora, can be used as an endogenous internal control nucleic acid in a method for detecting target microorganisms from a sample collected from the skin using nucleic acid amplification.
  • the nucleic acid detection rate of Corynebacterium tuberculostearicum was confirmed from samples collected from multiple human skin, and compared with the detection rates of Human beta globin (HBB) and Human Bacteroides spp. (HuBac) genes, which have been used as endogenous internal controls.
  • HBB Human beta globin
  • Human Bacteroides spp. Human Bacteroides spp.
  • a nucleic acid encoding 16s rRNA was used as the target nucleic acid of Corynebacterium tuberculostearicum (Table 1).
  • the detection of the nucleic acid was performed using TOCE TM technology (WO 2012/096523), which can detect multiple targets by utilizing signals generated by dimers formed depending on the presence of the target nucleic acid sequence.
  • primer pairs for detecting the target nucleic acid encoding the 16s rRNA of Corynebacterium tuberculostearicum are shown in Table 2.
  • HBB a primer pair for HBB detection (Allplex TM H. pylori & ClariR Assay product) and a PTO and CTO designed based on the primer pair were used.
  • HuBac a primer pair for HuBac detection (Allplex TM Entero-DR Assay product) and a PTO and CTO designed based on the primer pair were used.
  • Example 1-2 Corynebacterium tuberculostearicum Comparison of detection rates of HBB and HuBac
  • eSwab (Cat. No. 480CE; Copan) was used to collect skin samples from each site (2 axillary samples, 2 groin samples, 3 axillary and groin (mixed) samples).
  • Nucleic acid was extracted from the collected skin samples using the automated extraction equipment Seegene NIMBUS (Cat. No. 65415-03) and the extraction reagent STARMag 96X4 Universal Cartridge kit (Cat No. 744300.4.UC384). 300 ⁇ l of sample was used for nucleic acid extraction, and elution was performed in a volume of 100 ⁇ l. The obtained nucleic acid extract was used for real-time polymerase chain reaction.
  • Taq DNA polymerase with 5' nuclease activity was used for extension of forward and reverse primers, cleavage of PTO, and extension of CTO.
  • Corynebacterium tuberculostearicum on nucleic acid extracts obtained from the same sample To compare the detection rates of target nucleic acids and HBB genes and HuBac genes, three tubes containing 5 ⁇ l of the same nucleic acid extract were prepared.
  • the first tube (tube 1) contained the extract of Corynebacterium tuberculostearicum.
  • the same amount of oligonucleotide for detecting Corynebacterium tuberculostearicum (forward and reverse primers, PTO, CTO) as that for detecting HBB was added.
  • the HuBac oligonucleotide 8 pmoles of forward primer, 8 pmoles of reverse primer, 8 pmoles of PTO, 2 pmoles of CTO was added.
  • a reaction mixture was prepared by adding 5 ⁇ l of 4X enzyme mixture [finally 3.2 mM dNTPs, 14 mM MgCl 2 , and 4 U Taq DNA polymerase] and 5 ⁇ l of RNase-free water to each of three tubes to a final volume of 20 ⁇ l.
  • Real-time PCR was performed using the reaction mixture prepared above.
  • the tubes containing the reaction mixtures were placed in a real-time thermocycler (CFX96, Bio-Rad) and denatured at 95°C for 15 min, followed by 45 cycles of 95°C for 10 sec, 60°C for 15 sec, and 72°C for 10 sec. Signal detection was performed at 60°C for each cycle, and the results are summarized in Tables 3 to 7 below.
  • the detection rates of Corynebacterium tuberculostearicum for skin samples collected by site using eSwab TM were 100% for axillary samples, 100% for groin samples, and 100% for axillary and groin (mixed) samples.
  • the detection rates of HBB were 50% for axillary samples, 100% for groin samples, and 100% for axillary and groin (mixed) samples.
  • the detection rates of HuBac were 0% for axillary samples, 100% for groin samples, and 100% for axillary and groin (mixed) samples.
  • the average Ct value of Corynebacterium tuberculostearicum was 30.01 for axillary specimens, 30.55 for inguinal specimens, and 29.83 for axillary and inguinal (mixed) specimens.
  • HBB showed an average Ct value of 41.35 for axillary specimens, 35.62 for inguinal specimens, and 32.23 for axillary and inguinal (mixed) specimens.
  • HuBac showed no detection in axillary specimens, an average Ct value of 37.40 for inguinal specimens, and an average Ct value of 35.15 for axillary and inguinal (mixed) specimens, confirming that the average Ct value of Corynebacterium tuberculostearicum is lower than that of HBB and HuBac.
  • the above detection rate represents the ratio of the number of detected signals to the total number of samples.
  • Example 2 From a sample taken from the skin Cutibacterium acnes Check the detection rate
  • the present inventors confirmed that the nucleic acid of Cutibacterium acnes , a bacterium selected from normal skin flora, can be used as an endogenous internal control nucleic acid in a method for detecting target microorganisms from a sample collected from the skin using nucleic acid amplification.
  • a nucleic acid encoding DNA gyrase subunit A (gyrA) of Cutibacterium acnes was used as the target nucleic acid (Table 8).
  • the detection of the nucleic acid was performed using TOCE TM technology (WO 2012/096523), which can detect multiple targets by utilizing a signal generated by a dimer formed depending on the presence of the target nucleic acid sequence.
  • the sequences of the primer pairs, PTO (Probing and Tagging Oligonucleotide) and CTO (Capturing and Templating Oligonucleotide), for detecting the target nucleic acid encoding gyrA of Cutibacterium acnes are shown in Table 9.
  • Example 2-2 Cutibacterium acnes Check the detection rate
  • eSwab (Cat. No. 480CE; Copan) was used to collect skin samples from each site (3 axillary samples, 3 groin samples, 3 axillary and groin (mixed) samples).
  • Nucleic acid extraction was performed using the automated extraction equipment Seegene NIMBUS (Cat. No. 65415-03) and the extraction reagent STARMag 96X4 Universal Cartridge kit (Cat No. 744300.4.UC384). 300 ⁇ l of sample was used for nucleic acid extraction, and elution was performed in a volume of 100 ⁇ l. The obtained nucleic acid extract was used for real-time polymerase chain reaction.
  • Tubes containing 5 ⁇ l of target nucleic acid extract of Cutibacterium acnes were prepared for nucleic acid extracts obtained from the same sample.
  • Tube 1 contained Cutibacterium acnes.
  • 4 pmoles of the forward primer (Sequence 7), 4 pmoles of the reverse primer (Sequence 8), 3 pmoles of PTO (Sequence 9), and 2 pmoles of CTO (Sequence 10) were added.
  • the detection rates of Cutibacterium acnes for skin samples collected by site using eSwab TM were 100% for the axillary samples, 100% for the groin samples, and 100% for the axillary and groin (mixed) samples.
  • the average Ct value of Cutibacterium acnes for the axillary samples was 35.43
  • the average Ct value for the groin samples was 34.73
  • the average Ct value for the axillary and groin (mixed) samples was 32.89.
  • the present inventors detected Acinetobacter baumannii as a skin microorganism, and used the oligonucleotide included in the AllplexTM MDRO Assay product (Seegene Inc, Korea) as an oligonucleotide for detecting Acinetobacter baumannii nucleic acid, and used the same oligonucleotide as used in Example 1 as an oligonucleotide for detecting internal control nucleic acid.
  • Example 3-1 As an internal control Corynebacterium tuberculostearicum Use of
  • this experiment used skin samples previously confirmed to be positive for Acinetobacter baumannii using aerobic culture and MALDI-TOF mass spectrometry, which are recognized as standard methods for detecting drug-resistant microorganisms.
  • the samples were cultured for 24 hours at 37°C under aerobic conditions using CHROMagar Acinetobacter medium for the selective isolation and identification of Acinetobacter bacteria.
  • Extraction of nucleic acids from the collected skin samples was performed using an automated extraction device, Microlab NIMBUS IVD (Cat. No. 65415-02, Hamilton) and an extraction reagent, STARMag 96X4 Universal Cartridge Kit (Cat. No. 744300.4.UC384, Seegene Inc.). 300 ⁇ l of each sample was used for nucleic acid extraction, and elution was performed in a volume of 100 ⁇ l. The obtained nucleic acid extracts were used for real-time polymerase chain reaction.
  • Taq DNA polymerase with 5' nuclease activity was used for extension of forward and reverse primers, cleavage of PTO, and extension of CTO.
  • Corynebacterium tuberculostearicum nucleic acid can be useful as an endogenous internal control in the detection process of Acinetobacter baumannii.

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Abstract

La présente invention concerne un procédé de détection d'un acide nucléique d'un micro-organisme cible à partir d'un échantillon cutané, en utilisant un acide nucléique de Corynebacterium spp. ou Cutibacterium spp. comme acide nucléique témoin interne endogène, ainsi qu'une composition pour l'amplification d'acide nucléique utilisée dans ce procédé. Le témoin interne endogène selon la présente invention est présent dans l'échantillon dès le début, ce qui élimine l'inconvénient de devoir l'ajouter séparément après le prélèvement de l'échantillon, et peut être utilisé comme témoin interne pour le processus de prélèvement d'échantillons, comme témoin interne pour le processus d'extraction d'acide nucléique et comme témoin interne pour le processus d'amplification d'acide nucléique. De plus, en utilisant l'acide nucléique de Corynebacterium spp. ou Cutibacterium spp. comme témoin interne endogène, il est possible de détecter la présence ou l'absence d'un acide nucléique microbien cible dans un échantillon cutané prélevé avec une grande précision, en minimisant les faux négatifs et les faux positifs.
PCT/KR2025/008138 2024-06-27 2025-06-13 Procédé de détection d'acide nucléique d'un micro-organisme cible à partir d'un échantillon cutané à l'aide de corynebacterium spp. ou cutibacterium spp. comme témoin interne endogène Pending WO2026005351A1 (fr)

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US20150051082A1 (en) * 2011-12-22 2015-02-19 Animal Health Trust Diagnostic test for bacterial pathogens using internal control bacterial strain
US20220348986A1 (en) * 2019-06-19 2022-11-03 T2 Biosystems, Inc. Methods and compositions for comprehensive and high sensitivity detection of pathogens and drug resistance markers
WO2023027073A1 (fr) * 2021-08-27 2023-03-02 株式会社島津製作所 Procédé de pcr quantitative utilisant une commande interne
KR20230097143A (ko) * 2020-11-05 2023-06-30 백톤 디킨슨 앤드 컴퍼니 박테리아 호흡기 병원체의 멀티플렉스 검출
KR102575756B1 (ko) * 2018-05-10 2023-09-07 주식회사 씨젠 내부 대조군으로 장내 정상세균총을 이용하여 시료로부터 장내 미생물을 검출하는 방법

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US20150051082A1 (en) * 2011-12-22 2015-02-19 Animal Health Trust Diagnostic test for bacterial pathogens using internal control bacterial strain
KR102575756B1 (ko) * 2018-05-10 2023-09-07 주식회사 씨젠 내부 대조군으로 장내 정상세균총을 이용하여 시료로부터 장내 미생물을 검출하는 방법
US20220348986A1 (en) * 2019-06-19 2022-11-03 T2 Biosystems, Inc. Methods and compositions for comprehensive and high sensitivity detection of pathogens and drug resistance markers
KR20230097143A (ko) * 2020-11-05 2023-06-30 백톤 디킨슨 앤드 컴퍼니 박테리아 호흡기 병원체의 멀티플렉스 검출
WO2023027073A1 (fr) * 2021-08-27 2023-03-02 株式会社島津製作所 Procédé de pcr quantitative utilisant une commande interne

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AHMED NASHWA, JOGLEKAR PAYAL, DEMING CLAYTON, , LEMON KATHERINE P., KONG HEIDI H., SEGRE JULIE A., CONLAN SEAN, BARNABAS BEATRICE : "Genomic characterization of the i C. tuberculostearicum /i species complex, a prominent member of the human skin microbiome", MSYSTEMS, HIGHWIRE PRESS (FREE ACCESS), vol. 8, no. 6, 21 December 2023 (2023-12-21), XP093383680, ISSN: 2379-5077, DOI: 10.1128/msystems.00632-23 *
DATABASE Nucleotide 16 June 2021 (2021-06-16), ANONYMOUS: "Corynebacterium sp. strain JLM5 16S ribosomal RNA gene, partial sequence", XP093383685, Database accession no. MW979572.1 *

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