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WO2024143780A1 - Composition pour la détection d'un champignon dominant d'une infection articulaire périprothétique, et son utilisation - Google Patents

Composition pour la détection d'un champignon dominant d'une infection articulaire périprothétique, et son utilisation Download PDF

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Publication number
WO2024143780A1
WO2024143780A1 PCT/KR2023/014246 KR2023014246W WO2024143780A1 WO 2024143780 A1 WO2024143780 A1 WO 2024143780A1 KR 2023014246 W KR2023014246 W KR 2023014246W WO 2024143780 A1 WO2024143780 A1 WO 2024143780A1
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seq
base sequence
composition
detecting
fungal infection
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English (en)
Korean (ko)
Inventor
변의석
김성현
김동현
이지영
백은영
제원근
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Dreamdx Co Ltd
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Dreamdx Co Ltd
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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
    • C12Q1/6876—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
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the present invention relates to a composition for detecting dominant fungi in infections around artificial knee joint replacements and its use. More specifically, it relates to a composition for detecting only the dominant fungi among the dominant fungi and the dominant fungi in infections around artificial knee joint replacements; It relates to a kit using this and a detection method using it.
  • periprosthetic knee joint replacement infection PJI
  • intraoperative infection PJI
  • nosocomial infection include Staphylococcus epidermidis , Staphylococcus aureus , and Enterobacteriaceae.
  • Enterococcus faecalis Streptococcus dysgalactiae, Streptococcus pyogenes , Esherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Enterobacter cloacae
  • the dominant fungi include Candida parapsilosis , Candida tropicalis , Candida glabrata , and Candida albicans ( Candida albicans ), Aspergillus fumigatus ( Aspergillus fumigatus ), etc.
  • the 16S rRNA gene of bacteria and the 18S rRNA gene of fungi are commonly used in the classification of bacteria and fungi.
  • the genetic identity of the gene region is high between fungal species, so the discriminatory power is low. There is a problem with it falling. Therefore, there is a problem in that it is difficult to distinguish between the dominant bacterial and dominant fungal infections that cause infections around artificial knee joint replacements in the patient's synovial fluid.
  • the type of therapeutic agent administered varies depending on the type of infectious bacteria. For example, if the causative bacteria for periartificial knee joint replacement infection is bacteria, antibiotics are administered, and if the causative bacteria are fungi, antifungal drugs are administered. Identifying the type of causative bacteria is very important in the future treatment process for periartificial knee joint replacement infection. do. Therefore, there is a growing need for technology to identify the type of bacteria causing infections around artificial knee joint replacements.
  • the purpose of the present invention is to provide a composition for detecting fungal infections that can distinguish and detect only the dominant fungi among the dominant bacteria and dominant fungi in infections around artificial knee joint replacements.
  • Another object of the present invention is to provide a method for detecting predominant fungi in infections around artificial knee joint replacements using the composition for detecting fungal infections.
  • the present invention provides a forward primer base sequence of SEQ ID NO: 1 and a reverse primer base sequence of SEQ ID NO: 2; and a forward primer base sequence of SEQ ID NO: 4 and a reverse primer base sequence of SEQ ID NO: 5. It provides a composition for detecting fungal infection comprising at least one primer set selected from the group consisting of.
  • the present invention provides a kit for detecting fungal infection comprising the composition for detecting fungal infection.
  • the detection step uses real-time polymerase chain reaction (Real-time PCR) technology and is characterized by annealing at a temperature of 53 to 62°C.
  • Real-time PCR real-time polymerase chain reaction
  • Figure 1 is a diagram showing the results of multi-alignment comparison of the genes of Candida species, among the dominant fungal species in infections around artificial knee joint replacements, to design primers and probes of SEQ ID NOs: 1 to 3.
  • Figure 10 is a standard curve graph converting the amplification curve of Candida tropicalis according to Figure 5 into Log values.
  • the composition for detecting fungal infection of the present invention includes the forward primer base sequence of SEQ ID NO: 1, the reverse primer base sequence of SEQ ID NO: 2, the forward primer base sequence of SEQ ID NO: 4, and the reverse primer base sequence of SEQ ID NO: 5. can do.
  • the kit may be a kit that can distinguish and detect only the dominant fungi among the dominant bacteria and dominant fungi in infections around artificial knee joint replacements.
  • the kit includes next generation sequencing (NGS), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), Northern blotting, and RNase protection assay ( It may be used for RNase protection assay (RPA) or microarray chip, and preferably may be used for real-time polymerase chain reaction (Real-time PCR).
  • NGS next generation sequencing
  • RT-PCR reverse transcription polymerase chain reaction
  • Real-time PCR real-time polymerase chain reaction
  • Northern blotting and RNase protection assay
  • RNase protection assay RPA
  • RPA real-time polymerase chain reaction
  • the “test subject” may be an individual suspected of having an antibiotic-resistant bacterial infection, an individual showing symptoms of a disease or complication due to an antibiotic-resistant bacterial infection, etc.
  • the biological sample includes body fluid samples, including blood, serum, plasma, lymph, breast milk, urine, feces, ocular fluid, saliva, semen, brain extract (e.g., brain pulverized material), spinal fluid, appendix, Includes, but is not limited to, spleen and tonsil tissue extracts.
  • body fluid samples including blood, serum, plasma, lymph, breast milk, urine, feces, ocular fluid, saliva, semen, brain extract (e.g., brain pulverized material), spinal fluid, appendix, Includes, but is not limited to, spleen and tonsil tissue extracts.
  • the detection methods include real-time polymerase chain reaction (Real-time PCR), DNA sequencing, hybridization by microarray, PCR-RFLP (Restriction fragment length polymorphism) analysis, and RAPD (Randomly amplified polymorphic DNA).
  • Real-time PCR real-time polymerase chain reaction
  • DNA sequencing DNA sequencing
  • hybridization by microarray PCR-RFLP (Restriction fragment length polymorphism) analysis
  • RAPD Randomly amplified polymorphic DNA
  • the method for detecting predominant fungal infections around artificial knee joint replacements using real-time PCR detects fungal infections comprising the forward primer base sequence of SEQ ID NO: 1, the reverse primer base sequence of SEQ ID NO: 2, and the probe base sequence of SEQ ID NO: 3.
  • Preparing a PCR composition by mixing the biological sample of the test subject with the composition; And performing real-time PCR using the mixture to detect predominant fungal infection around the artificial knee joint replacement.
  • the PCR composition includes 0.1 to 2 parts by weight of the forward primer of SEQ ID NO: 1, based on 20 parts by weight of the total PCR composition; 0.1 to 2 parts by weight of reverse primer of SEQ ID NO: 2; 0.1 to 2 parts by weight of the probe of SEQ ID NO: 3; and 1 to 3 parts by weight of a biological sample of the test subject; preferably, 0.5 to 1.5 parts by weight of the forward primer of SEQ ID NO: 1 based on 20 parts by weight of the total PCR composition; 0.5 to 1.5 parts by weight of reverse primer of SEQ ID NO: 2; 0.5 to 1.5 parts by weight of the probe of SEQ ID NO: 3; and 1.5 to 2.5 parts by weight of template DNA.
  • the forward primer of SEQ ID NO: 1 can be used at a concentration of 2 to 6 ⁇ M, preferably at a concentration of 3 to 5 ⁇ M.
  • the reverse primer of SEQ ID NO: 2 can be used at a concentration of 2 to 6 ⁇ M, and preferably at a concentration of 3 to 5 ⁇ M.
  • the probe of SEQ ID NO: 3 can be used at a concentration of 0.1 to 4 ⁇ M, preferably 1 to 2 ⁇ M.
  • the method for detecting predominant fungal infections around artificial knee joint replacements using the real-time PCR is a fungal infection comprising the forward primer base sequence of SEQ ID NO: 4, the reverse primer base sequence of SEQ ID NO: 5, and the probe base sequence of SEQ ID NO: 6.
  • Preparing a PCR composition by mixing a biological sample of a test subject with a composition for detecting infection; And performing real-time PCR using the mixture to detect predominant fungal infection around the artificial knee joint replacement.
  • the PCR composition includes 0.1 to 2 parts by weight of the forward primer of SEQ ID NO: 4 based on 20 parts by weight of the total PCR composition; 0.1 to 2 parts by weight of reverse primer of SEQ ID NO: 5; 0.1 to 2 parts by weight of the probe of SEQ ID NO: 6; and 1 to 3 parts by weight of a biological sample of the test subject; preferably, 0.5 to 1.5 parts by weight of the forward primer of SEQ ID NO: 4 based on 20 parts by weight of the total PCR composition; 0.5 to 1.5 parts by weight of reverse primer of SEQ ID NO: 5; 0.5 to 1.5 parts by weight of the probe of SEQ ID NO: 6; and 1.5 to 2.5 parts by weight of a biological sample from the test subject.
  • the method for detecting predominant fungal infections around artificial knee joint replacements using real-time PCR includes the forward primer base sequence of SEQ ID NO: 1, the reverse primer base sequence of SEQ ID NO: 2, the probe base sequence of SEQ ID NO: 3, and SEQ ID NO: 4.
  • Preparing a PCR composition by mixing a biological sample of a test subject with a composition for detecting fungal infection containing the forward primer base sequence, the reverse primer base sequence of SEQ ID NO: 5, and the probe base sequence of SEQ ID NO: 6; And performing real-time PCR using the mixture to detect predominant fungal infection around the artificial knee joint replacement.
  • the forward primers of SEQ ID NOs: 1 and 4 can be used at a concentration of 2 to 6 ⁇ M, preferably at a concentration of 3 to 5 ⁇ M.
  • the reverse primers of SEQ ID NOs: 2 and 5 can be used at a concentration of 2 to 6 ⁇ M, and preferably at a concentration of 3 to 5 ⁇ M.
  • the probes of SEQ ID NOs: 3 and 6 can be used at a concentration of 0.1 to 4 ⁇ M, preferably 1 to 2 ⁇ M.
  • Real-time polymerase chain reaction (Real-time PCR) using the PCR composition includes the steps of denaturing for 10 to 20 seconds at a temperature of 90 to 100 ° C.; Annealing at a temperature of 53 to 62°C for 20 to 40 seconds; and elongation at a temperature of 67 to 77°C for 20 to 40 seconds to amplify the target gene, preferably at a temperature of 93 to 97°C for 10 to 20 seconds. step; Annealing at a temperature of 56 to 60°C for 20 to 40 seconds; and elongation at a temperature of 70 to 74°C for 20 to 40 seconds.
  • the target gene can be amplified by repeating the steps. The repetition may be repeated for 35 to 45 cycles.
  • the target gene may not be sufficiently amplified and the target gene may not be detected, and if the repetition number is more than 45 cycles, the target gene may not be detected. If this is done, there may be a problem that the detection time takes too long.
  • Example 1 was performed to create a primer set that could specifically detect the target region present in 18s rRNA in the fungus that causes infection around artificial knee joint replacement.
  • the 18s rRNA sequences of four Candida species among the five dominant fungal species causing infections around artificial knee joint replacements were collected from GeneBank (www.ncbi.nlm.nih.gov), and the sequences were compared using multi-alignment.
  • the four species of Candida include Candida parapsilosis, Candida tropicalis, Candida glabrata , and Candida albicans.
  • primer sequences of SEQ ID NOs: 1 and 2 were designed from sequences with almost no mismatches.
  • the multi-alignment results of the four Candida species are shown in Figure 1, and the primer base sequences of SEQ ID NOs: 1 and 2 are shown in Table 1.
  • the 18s rRNA sequence of one Aspergillus species was collected from GeneBank (www.ncbi.nlm.nih.gov) and the sequences were compared using multi-alignment. did.
  • the Aspergillus species refers to Aspergillus fumigatus .
  • primer sequences of SEQ ID NOs: 4 and 5 were designed from sequences with almost no mismatches.
  • the multi-alignment results of the Aspergillus species are shown in Figure 2, and the primer base sequences of SEQ ID NOs: 4 and 5 are shown in Table 1.
  • Example 2 Production of a probe for detecting predominant fungal infections around artificial knee joint replacements.
  • Example 2 a probe was prepared to perform TaqMan probe real-time polymerase chain reaction (Real-time PCR) using the primers prepared in Example 1.
  • the 18s rRNA sequences of the four Candida species were multi-aligned and compared in the same manner as in Example 1, and the probe base sequence of SEQ ID NO: 3 was designed from sequences with almost no mismatches.
  • the 5' end of the probe base sequence of SEQ ID NO: 3 was labeled with TET (Tetrachlorofuroscein) fluorescent substance, and the 3' end was labeled with TAMRA (5-Carboxytetramethylrhodamine) quencher.
  • TET Tetrachlorofuroscein
  • TAMRA 5-Carboxytetramethylrhodamine
  • the 18s rRNA sequences of one Aspergillus species were compared by multi-alignment in the same manner as in Example 1, and the probe base sequence of SEQ ID NO: 6 was designed from the sequence with almost no mismatch.
  • the 5' end of the probe base sequence of SEQ ID NO: 6 was labeled with 6-FAM (6-Carboxyfluorescein) fluorescent substance, and the 3' end was labeled with TAMRA (5-Carboxytetramethylrhodamine) quencher.
  • 6-FAM 6-Carboxyfluorescein
  • TAMRA 5-Carboxytetramethylrhodamine
  • the primer set containing the forward primer base sequence of SEQ ID NO: 1, the reverse primer base sequence of SEQ ID NO: 2, and the probe base sequence of SEQ ID NO: 3 prepared in Examples 1 and 2 are the dominant fungal species for infections around artificial knee joint replacements.
  • four species of Candida can be detected, and the primer set including the forward primer base sequence of SEQ ID NO: 4, the reverse primer base sequence of SEQ ID NO: 5, and the probe base sequence of SEQ ID NO: 6 is the forward primer base of SEQ ID NO: 1.
  • a primer set containing the sequence, reverse primer base sequence of SEQ ID NO: 2, and probe base sequence of SEQ ID NO: 3 can detect Aspergillus species.
  • the four species of Candida include Candida parapsilosis, Candida tropicalis, Candida glabrata , and Candida albicans .
  • Example 3 Predominant fungal infection detection test using primers and probes for detecting predominant fungal infections surrounding artificial knee replacement implants
  • Example 3 was performed to detect the dominant fungal infection using primers and probes for detecting the dominant fungal infection surrounding artificial knee joint replacements produced in Examples 1 and 2.
  • the standard strains of dominant fungi used in Example 3 were Candida parapsilosis (KCTC7653 Candida parapsilosis ), Candida tropicalis (KCTC7212 Candida tropicalis ), Candida glabrata (KCTC7219 Candida glabrata ), and Candida albicans (ATCC 10231 Candida) . albicans ) and Aspergillus fumigatus (KCTC6145 Aspergillus fumigatus ) were used.
  • Sequencing was performed to confirm whether the genes amplified in FIG. 3 correspond to the four standard strains of Candida species among the dominant fungi used as templates.
  • ITS sequencing primer
  • the sequences of four Candida species among the dominant fungi were sequenced in both primers, which means that the primers and probes produced in Examples 1 and 2 were used to sequence the four Candida species among the dominant fungi. It means accurately amplified.
  • the sequencing results using the two primers above are listed in Table 5.
  • ITS sequencing primer
  • the sequence of Aspergillus fumigatus among the dominant fungi was sequenced in both primers, which means that the primers and probes prepared in Examples 1 and 2 were It means an accurate amplification of Aspergillus fumigatus , one of the dominant fungi.
  • the sequencing results using the above two primers are listed in Table 6.
  • Experimental Example 1 was conducted to evaluate the sensitivity of five dominant types of fungi, which are bacteria causing infections around artificial knee joint replacements, using the primers and probes prepared in Examples 1 and 2 above.
  • genes for detection of the five dominant species of fungi were synthesized, and the genes for detection were synthesized as part of the 18s rRNA gene of the fungus.
  • the sequences of the synthesized fungal genes are shown in SEQ ID NOs: 7 to 11.
  • the SEQ ID NOs: 7 to 11 are sequences of Candida parapsilosis, Candida tropicalis, Candida glabrata, Candida albicans, or Aspergillus fumigatus, in that order.
  • Experimental Example 2 was conducted to evaluate the specificity of the five dominant fungal species that cause infections around artificial knee joint replacements using the primers and probes prepared in Examples 1 and 2 above. Genes for detection of the five dominant fungal species were synthesized in the same manner as in Experimental Example 1. Real-time PCR compositions and conditions performed in Experimental Example 2 were the same as those in Tables 2 to 4, and the template DNA was Synthetic genes from five dominant fungal species were used.
  • Experimental Example 3-1 was conducted to determine whether only dominant fungi, not dominant bacteria, were detected using only the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 prepared in Example 1.

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Abstract

La présente invention concerne une composition permettant de détecter un champignon dominant d'une infection articulaire périprothétique, et son utilisation, et plus particulièrement : une composition pouvant distinguer et détecter uniquement un champignon dominant entre une bactérie dominante et le champignon dominant d'une infection articulaire périprothétique ; un kit l'utilisant ; et un procédé de détection l'utilisant.
PCT/KR2023/014246 2022-12-29 2023-09-20 Composition pour la détection d'un champignon dominant d'une infection articulaire périprothétique, et son utilisation Ceased WO2024143780A1 (fr)

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KR10-2022-0189917 2022-12-29
KR1020220189917A KR102546048B1 (ko) 2022-12-29 2022-12-29 진균 감염 검출용 조성물 및 이를 이용한 검출방법

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KR102546048B1 (ko) * 2022-12-29 2023-06-23 드림디엑스 주식회사 진균 감염 검출용 조성물 및 이를 이용한 검출방법

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KR20200009054A (ko) * 2017-05-17 2020-01-29 마이크로바이오 피티와이 엘티디 바이오마커 및 그의 용도
KR20220073327A (ko) * 2020-11-26 2022-06-03 고려대학교 산학협력단 형광 프로브를 이용한 칸디다 알비칸, 칸디다 아우리스, 및 칸디다 글라브라타를 감별하여 검출할 수 있는 동시다중 등온증폭반응 프라이머 세트
KR102546048B1 (ko) * 2022-12-29 2023-06-23 드림디엑스 주식회사 진균 감염 검출용 조성물 및 이를 이용한 검출방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20150007054A (ko) * 2013-07-10 2015-01-20 솔젠트 (주) 중합효소연쇄반응 및 실시간중합효소연쇄반응을 이용한 세포치료제 또는 생물학적 의약품에 오염되는 진균을 검출하는 방법 및 이 방법에 사용하기 위한 키트
KR101521409B1 (ko) * 2015-01-15 2015-05-18 경북대학교 산학협력단 진균성 및 아메바성 각막염의 원인체를 동시에 검출할 수 있는 다중 중합효소 연쇄반응 프라이머 세트 및 이를 이용한 각막염 유발균 검출 방법
KR20200009054A (ko) * 2017-05-17 2020-01-29 마이크로바이오 피티와이 엘티디 바이오마커 및 그의 용도
KR20220073327A (ko) * 2020-11-26 2022-06-03 고려대학교 산학협력단 형광 프로브를 이용한 칸디다 알비칸, 칸디다 아우리스, 및 칸디다 글라브라타를 감별하여 검출할 수 있는 동시다중 등온증폭반응 프라이머 세트
KR102546048B1 (ko) * 2022-12-29 2023-06-23 드림디엑스 주식회사 진균 감염 검출용 조성물 및 이를 이용한 검출방법

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