KR100440746B1 - Genotyping kit for Selecting Antibiotics Resistant Bacteria - Google Patents
Genotyping kit for Selecting Antibiotics Resistant Bacteria Download PDFInfo
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- KR100440746B1 KR100440746B1 KR10-2001-0005995A KR20010005995A KR100440746B1 KR 100440746 B1 KR100440746 B1 KR 100440746B1 KR 20010005995 A KR20010005995 A KR 20010005995A KR 100440746 B1 KR100440746 B1 KR 100440746B1
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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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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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
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
본 발명은 베타-락탐계 항생물질에 대한 내성을 가지는 균주를 선별하기 위한 유전형 분석키트 및 전기 분석키트를 사용하여 베타-락탐계 항생물질에 대한 내성을 가지는 균주를 선별하는 방법에 관한 것이다. 본 발명의 베타-락탐계 항생제 내성균을 선별하기 위한 유전형 분석키트는 베타-락타메이즈 유전자와 상보적으로 결합할 수 있는 탐침을 포함하는 DNA칩, 시료로부터 채취한 DNA를 PCR방법으로 증폭시키기 위한 프라이머 및 전기 DNA칩과 보합반응되는 증폭된 DNA를 탐지하기 위한 표지수단인 Cy5-dCTP를 포함하고, 전기 분석키트를 이용하여 베타-락탐계 항생제 내성균을 선별하는 방법은 유전형 분석키트의 프라이머를 사용하여 검사할 시료로부터 채취한 DNA를 증폭시키는 단계; 전기 증폭된 DNA를 DNA칩에 적용하여, 증폭된 DNA와 탐침을 보합반응시키는 단계; 및, 탐침과 보합반응된 DNA를 검출하는 단계를 포함한다. 본 발명의 유전형 분석키트를 이용하면, 간단한 방법으로 항생제에 내성을 가지는 병원성 균주를 탐지할 수 있으므로, 병원성 세균의 연구에 널리 활용될 수 있을 것이다.The present invention relates to a method for screening strains resistant to beta-lactam antibiotics using genotyping kits and electroanalytical kits for screening strains resistant to beta-lactam antibiotics. The genotyping kit for selecting beta-lactam antibiotic resistant bacteria of the present invention is a DNA chip comprising a probe capable of complementarily binding to a beta-lactamase gene, and a primer for amplifying DNA obtained from a sample by PCR. And Cy5-dCTP, which is a labeling means for detecting amplified DNA that reacts with the electric DNA chip. The method for selecting beta-lactam antibiotic resistance bacteria using an electric analysis kit is performed by using a primer of a genotyping assay kit. Amplifying the DNA collected from the sample to be tested; Applying the electrically amplified DNA to the DNA chip to perform a hybrid reaction between the amplified DNA and the probe; And detecting DNA that has been reacted with the probe. Using the genotyping kit of the present invention, it is possible to detect pathogenic strains resistant to antibiotics in a simple way, it will be widely used in the study of pathogenic bacteria.
Description
본 발명은 항생물질 내성균 선별을 위한 유전형 분석키트에 관한 것이다. 좀 더 구체적으로, 본 발명은 베타-락탐계 항생물질에 대한 내성을 가지는 균주를 선별하기 위한 유전형 분석키트 및 전기 분석키트를 사용하여 베타-락탐계 항생물질에 대한 내성을 가지는 균주를 선별하는 방법에 관한 것이다.The present invention relates to a genotyping kit for screening antibiotic resistant bacteria. More specifically, the present invention provides a method for screening strains resistant to beta-lactam antibiotics using genotyping kits and electroanalytical kits for screening strains resistant to beta-lactam antibiotics. It is about.
1928년에 플레밍(Fleming)이 페니실린을 발견한 이래 항생제는 감염 질환을 가진 수많은 사람들의 생명을 구하여 기적의 약으로 불려왔으나, 1960년대에 페니실린에 대한 내성균이 출현한 것을 필두로 여러 항생제가 개발, 사용됨에 따라, 그에 대한 내성균이 발생하는 문제점이 대두하게 되었다. 즉, 모든 미생물과 마찬가지로 병원균은 자기방어수단으로 항생제에 대한 내성을 돌연변이나 항생제 내성 유전자를 습득함으로써 얻게되는데, 이러한 내성균의 발현 빈도는 항생제의 오용과 남용이 많아짐에 따라 더욱 증가하고 있다.Since Fleming's discovery of penicillin in 1928, antibiotics have been called miracle drugs to save the lives of millions of people with infectious diseases, but several antibiotics have been developed since the emergence of penicillin-resistant bacteria in the 1960s. As it is used, a problem arises in which resistant bacteria develop. That is, as with all microorganisms, pathogens are obtained by obtaining mutation resistance or antibiotic resistance genes by self-defense, and the frequency of expression of these resistant bacteria is increasing as the abuse and abuse of antibiotics increases.
항생제 중 대표적인 것은 베타-락탐계 항생제로 이에 대한 내성은 이 항생제의 베타-락탐(β-lactam) 구조를 분해하는 효소인 베타-락타메이즈(β-lactamase)의 생성이 중요한 기전 중의 하나이다. 이 효소는 크게 네 가지로 분류되는데, 1 그룹은 클라불라닌산(clavulanic acid)에 의해 저해되지 않은 세팔로스포리나제(cephalosporinase), 2 그룹은 클라불라닌산에 의해 저해되는 페니실리나제(penicillinase)나 세팔로스포리나제, 3 그룹은 메탈로엔자임(metalloenzyme)이고, 4 그룹은 클라불라닌산에 의해 저해되지 않는 페니실리나제이다(참조: Bush K., Characterization of β-lactamase, Antimicrob. Agents Chemother., 33:259-265, 1989). 이들은 플라스미드에 의해 조절되는 것과염색체에 의해 조절되는 것이 있다.The most representative of the antibiotics is beta-lactam antibiotics, which is one of the important mechanisms for the production of beta-lactamase, an enzyme that breaks down the beta-lactam structure of the antibiotic. There are four main types of enzymes: group 1, cephalosporinase, which is not inhibited by clavulanic acid, and group 2, penicillinase, which is inhibited by clavulanic acid. B cephalosporins, group 3 is metalloenzyme, group 4 is penicillinase that is not inhibited by clavulanic acid (see Bush K., Characterization of β-lactamase, Antimicrob.Agents Chemother) , 33: 259-265, 1989). These are regulated by plasmids and by chromosomes.
지금까지 병원균이 내성을 갖고 있는 지를 검사하는 방법에는, 바우어-커비 테스트(Bauer-Kirby test), MIC(minimal inhibitory concentration), MBC(minimal bactericidal concentration), Serum killing power 또는 PCR법이 이용되고 있다(참조: 이연희, 항생제 내성-항생제 내성의 제문제, 미생물과 산업, 24:3-9, 1998). 디스크 검사법이라고도 불리는 바우어-커비 테스트는 뮬러힌톤(Muller Hinton) 한천 배지위에 세균이 있는 배양액을 접종한 후, 항생제가 들어있는 디스크를 놓아 그 주위에 생기는 억제환의 크기로 항생제에 대한 세균의 민감성을 측정하는 방법이다. MIC는 항생제가 연속적으로 희석된 유리관에 일정한 양의 세균을 넣고 성장여부를 결정하여, 박테리아의 성장을 억제하기 위해 필요한 최소한의 항생제 농도를 측정하는 것이다. MBC는 세균 배양액 중에 살아있는 박테리아의 99.9%를 죽이는 항생제의 농도를 의미한다. Serum killing power는 환자에 항생제를 투여한 후, 일정기간 후에 채취한 혈액이 박테리아 성장을 얼마나 억제하는 지를 측정한다. PCR은 내성유전자에 특이적으로 결합하는 프라이머를 사용하여 효소중합반응이 일어나는 지의 여부로 내성유전자의 존재 여부를 결정한다. 그러나, 종래의 항성제 내성균 탐지 방법들은 시간이 많이 걸릴 뿐만 아니라, 사용 방법이 복잡하여 한번에 다량의 시료를 신속하게 검사하는 데 큰 어려움이 있었다.Until now, the Bauer-Kirby test, MIC (minimal inhibitory concentration), MBC (minimal bactericidal concentration), Serum killing power, or PCR methods have been used to test whether the pathogen is resistant. See, Yeon-Hee Lee, Antibiotic Resistance-Problem of Antibiotic Resistance, Microorganisms and Industry, 24: 3-9, 1998). The Bauer-Kerby test, also called a disk test, inoculates a culture with bacteria on Muller Hinton agar media, then places a disk containing antibiotics to measure the sensitivity of the bacteria to antibiotics by the size of the inhibitor ring around it. That's how. MIC measures the minimum concentration of antibiotics needed to inhibit the growth of bacteria by placing a certain amount of bacteria in a glass tube in which the antibiotics are serially diluted. MBC refers to the concentration of antibiotics that kill 99.9% of live bacteria in bacterial culture. Serum killing power measures how much blood collected after a certain period of time inhibits bacterial growth after the patient is given antibiotics. PCR uses primers that specifically bind to a resistance gene to determine the presence of the resistance gene by whether the enzymatic polymerization reaction occurs. However, conventional methods for detecting antibiotic-resistant bacteria are not only time-consuming, but also have difficulty in quickly inspecting a large amount of samples at one time due to the complicated method of use.
따라서, 간단한 방법으로 다량의 시료를 신속하게 검사하여 항생제에 내성을 가지는 균주를 선별할 수 있는 방법을 개발하여야 할 필요성이 끊임없이 대두되었다.Therefore, there is a constant need to develop a method capable of quickly examining a large amount of samples by a simple method and selecting a strain resistant to antibiotics.
이에, 본 발명자들은 간단한 방법으로 다량의 시료를 신속하게 검사하여 항생제에 내성을 가지는 균주를 선별할 수 있는 방법을 개발하고자 예의 연구 노력한 결과, 베타-락타메이즈 유전자와 상보적으로 결합할 수 있는 탐침을 포함하는 DNA칩, 시료로부터 채취한 DNA를 PCR방법으로 증폭시키기 위한 프라이머 및 전기 DNA칩과 보합반응되는 증폭된 DNA를 탐지하기 위한 표지수단인 Cy5-dCTP를 포함하는 유전형 분석키트를 제작하고, 전기 분석키트를 사용하여 베타-락탐계 항생제에 대한 내성을 가지는 균주를 손쉽게 선별할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Thus, the present inventors have made a thorough research to develop a method that can quickly screen a large amount of samples by a simple method to select strains resistant to antibiotics, a probe that can be complementary to the beta-lactamase gene To prepare a genotyping kit comprising a DNA chip, a primer for amplifying the DNA collected from the sample by a PCR method, and Cy5-dCTP as a label means for detecting the amplified DNA that is conjugated with the electric DNA chip, It was confirmed that strains having resistance to beta-lactam antibiotics can be easily selected using the electric assay kit, and the present invention was completed.
결국, 본 발명은 주된 목적은 베타-락탐계 항생제 내성균을 선별하기 위한 유전형 분석키트를 제공하는 것이다.After all, the main object of the present invention is to provide a genotyping kit for screening beta-lactam antibiotic resistant bacteria.
본 발명의 다른 목적은 전기 유전형 분석키트에 포함된 DNA칩의 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing a DNA chip included in the electro genotyping kit.
본 발명의 또 다른 목적은 전기 분석키트를 이용하여 베타-락탐계 항생제 내성균을 선별하는 방법을 제공하는 것이다.Still another object of the present invention is to provide a method for selecting beta-lactam antibiotic resistant bacteria by using an assay kit.
도 1은 본 발명의 DNA 칩을 사용하여 OXA-1의 DNA를 검출한 결과를 나타내는 사진이다.1 is a photograph showing the results of detecting the DNA of OXA-1 using the DNA chip of the present invention.
도 2는 본 발명의 DNA 칩을 사용하여 MEN-1의 DNA를 검출한 결과를 나타내는 사진이다.Figure 2 is a photograph showing the results of detecting the DNA of MEN-1 using the DNA chip of the present invention.
도 3은 본 발명의 DNA 칩을 사용하여 SHV-1의 DNA를 검출한 결과를 나타내는 사진이다.Figure 3 is a photograph showing the results of detecting the DNA of SHV-1 using the DNA chip of the present invention.
도 4는 본 발명의 DNA 칩을 사용하여 OXY-1의 DNA를 검출한 결과를 나타내는 사진이다.4 is a photograph showing a result of detecting DNA of OXY-1 using the DNA chip of the present invention.
도 5는 본 발명의 DNA 칩을 사용하여 AmpC의 DNA를 검출한 결과를 나타내는 사진이다.5 is a photograph showing the results of detecting the DNA of AmpC using the DNA chip of the present invention.
본 발명의 베타-락탐계 항생제 내성균을 선별하기 위한 유전형 분석키트는베타-락타메이즈 유전자와 상보적으로 결합할 수 있는 탐침을 포함하는 DNA칩, 시료로부터 채취한 DNA를 PCR방법으로 증폭시키기 위한 프라이머 및 전기 DNA칩과 보합반응되는 증폭된 DNA를 탐지하기 위한 표지수단인 Cy5-dCTP를 포함한다: 이때, DNA칩은 탐침의 위치를 알려주는 기준 마커(marker)를 추가로 포함할 수도 있고, 표지수단이 특별히 제한되는 것은 아니나, 형광물질인 Cy5-dCTP를 사용함이 바람직하다. 아울러, 양성 대조군 및 음성 대조군을 사용할 수도 있는데, 양성 대조군으로는 인간의 TRAIL 수용체(human TRAIL receptor 2)를 사용함이 바람직하고, 음성 대조군으로는 인간의 종양괴사인자 수용체 결합인자(human TNF receptor associated factor)를 사용함이 바람직하다.The genotyping kit for selecting beta-lactam antibiotic resistant bacteria of the present invention is a DNA chip comprising a probe capable of complementarily binding to a beta-lactamase gene, and a primer for amplifying DNA obtained from a sample by PCR. And Cy5-dCTP, which is a labeling means for detecting amplified DNA that reacts with the electric DNA chip, wherein the DNA chip may further include a reference marker indicating the position of the probe. The means is not particularly limited, but it is preferable to use Cy5-dCTP which is a fluorescent material. In addition, it is also possible to use a positive control and a negative control, it is preferable to use the human TRAIL receptor (human TRAIL receptor 2) as a positive control, the human TNF receptor associated factor (human TNF receptor associated factor as a negative control) Is preferably used.
한편, 전기 유전형 분석키트에 포함된 DNA칩의 제조방법은 베타-락타메이즈 DNA와 상보적으로 결합할 수 있는 염기서열의 5'말단에 아민이 결합된 DNA 탐침을 작제하는 공정; 전기 작제된 DNA 탐침을 알데히드가 결합된 고체의 표면에 시프염기반응(Schiff's base reaction)을 통해 결합시키는 공정; 및, 전기 DNA 탐침이 결합된 고체표면에 아민과 결합하지 않고 남아 있는 알데히드를 환원시키는 공정을 포함한다.On the other hand, the method of manufacturing a DNA chip included in the electro-genotyping kit comprises the steps of constructing a DNA probe in which the amine is bound to the 5 'end of the base sequence that can be complementary to beta-lactamase DNA; Binding an electrically constructed DNA probe to a surface of an aldehyde-bound solid through a Schiff's base reaction; And reducing the aldehyde remaining without binding to the amine on the solid surface to which the electric DNA probe is bound.
이하, 본 발명의 DNA칩의 제조방법을 공정별로 나누어 보다 구체적으로 설명하고자 한다.Hereinafter, the method of manufacturing the DNA chip of the present invention will be described in more detail by dividing the process.
제 1공정: 탐침의 작제 Step 1 : constructing the probe
베타-락타메이즈 DNA와 상보적으로 결합할 수 있는 염기서열의 5'말단에 아민이 결합된 DNA 탐침을 작제한다: 이때, 탐침은 베타-락타메이즈 DNA의 염기서열을 갖도록 설계 및 합성되고, 합성된 염기서열이 고체의 표면에 결합될 수 있도록 염기서열의 5'말단에 결합시켜서 탐침을 작제한다.A DNA probe with an amine bound to the 5 'end of the base sequence capable of complementarily binding to the beta-lactamase DNA is constructed: The probe is designed, synthesized, and synthesized to have the base sequence of the beta-lactamase DNA. The probe is constructed by binding to the 5 ′ end of the base sequence so that the sequence can be bound to the surface of the solid.
제 2공정: 탐침의 고정화 Second Step : Immobilization of the Probe
전기 작제된 DNA 탐침을 알데히드가 결합된 고체의 표면에 시프염기반응(Schiff's base reaction)을 통해 결합시킨다: 이때, 고체는 특별히 제한되는 것은 아니나, 유리인 것이 바람직하다. 이때, 탐침의 농도는 100 내지 300pmol/ml가 바람직하고, 가장 바람직하게는 200pmol/ml이다.The electrically constructed DNA probe is bound to the surface of the aldehyde-bound solid via a Schiff's base reaction: the solid is not particularly limited but is preferably glass. At this time, the concentration of the probe is preferably 100 to 300 pmol / ml, most preferably 200 pmol / ml.
제 3공정: DNA칩의 제조 Third Step : Manufacture of DNA Chip
전기 DNA 탐침이 결합된 고체표면에 아민과 결합하지 않고 남아있는 알데히드를 환원시켜서, DNA칩을 제조한다: 이때, 알데히드의 환원조건이 특별히 제한되는 것은 아니나, NaBH4등의 환원제를 사용함이 바람직하다.The DNA chip is prepared by reducing the aldehyde remaining without binding to the amine on the solid surface to which the electric DNA probe is bound. At this time, the reducing conditions of the aldehyde are not particularly limited, but it is preferable to use a reducing agent such as NaBH 4 . .
전기방법으로 제조된 DNA칩을 포함하는 베타-락탐계 항생제 내성균을 선별하기 위한 유전형 분석키트를 이용하여 베타-락탐계 항생제 내성균을 선별하는 방법은 유전형 분석키트의 프라이머를 사용하여 검사할 시료로부터 채취한 DNA를 증폭시키는 단계; 전기 증폭된 DNA를 DNA칩에 적용하여, 증폭된 DNA와 탐침을 보합반응시키는 단계; 및, 탐침과 보합반응된 DNA를 검출하는 단계를 포함한다. 이때, 증폭된 시료가 표지자를 포함하기 위하여, Cy5-dCTP를 사용한 중합효소연쇄반응(PCR)을 통하여 증폭시료를 수득한다.The method of screening for beta-lactam antibiotic resistant bacteria using a genotyping kit for screening beta-lactam antibiotic resistant bacteria containing a DNA chip prepared by the above method is performed by using a primer of the genotyping kit from a sample to be tested. Amplifying the DNA; Applying the electrically amplified DNA to the DNA chip to perform a hybrid reaction between the amplified DNA and the probe; And detecting DNA that has been reacted with the probe. At this time, in order to amplify the sample to include a marker, an amplified sample is obtained through polymerase chain reaction (PCR) using Cy5-dCTP.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예 1: DNA칩의 제조 Example 1 Preparation of DNA Chip
베타-락탐계 항생제에 대한 내성 유전자를 사용하여 5'말단에 아민(amine)기가 포함되도록 각각의 탐침을 작제하였다. 각 탐침은 다음의 프라이머들을 사용하여 PCR로 증폭하여 제작하였다. 각 내성 유전자들을 증폭하기 위한 프라이머들의 염기서열은 다음과 같다: 프라이머 TEMf: 5'-TCGGGGAAATGTGCGCG-3'(서열번호 1),프라이머 TEMr: 5'-TGCTTAATCAGTGAGGCACC-3'(서열번호 2), 프라이머 SHV-1f: 5'-AAGATCCACTATCGCCAGGAGG-3'(서열번호 3), 프라이머 SHV-1r: 5'-ATTCAGTTCGTTTCCCAGCGG-3'(서열번호 4), 프라이머 MEN-1f: 5'-TCCTCTCTTCCAGA-3'(서열번호 5), 프라이머 MEN-1r: 5'-CAGCGCTTTTCGCGTCTAAG-3'(서열번호 6), 프라이머 CMY-1f: 5'-ATGCAACAACGACAATCCATC-3'(서열번호 7), 프라이머 CMY-1r: 5'-GTTGGGTAGTTGCGATTGG-3'(서열번호 8), 프라이머 OXY-1f: 5'-CAGATCTCGAGAAGCGTTCA-3'(서열번호 9), 프라이머 OXY-1r: 5'-ACCTCTTTGCGGTTTTTCGC-3'(서열번호 10), 프라이머 FOXf: 5'-CACCACGAGAATAACCAT-3'(서열번호 11), 프라이머 FOXr: 5'-ATGTGGACGCCTTGAACT-3'(서열번호 12), 프라이머 PSE-1f: 5'-AATGGCATTCAGCGCTTCCC-3'(서열번호 13), 프라이머 PSE-1r: 5'-GGGGCTTGATGCTCACTC C-3'(서열번호 14), 프라이머 OXA-1f: 5'-TCAACTTTCAAGATCGCA-3'(서열번호 15), 프라이머 OXA-1r: 5'-GTGTGTTTAGAATGGTGA-3'(서열번호 16), 프라이머 AmpCf: 5'-CTACGGTCTGGCTGCTA-3'(서열번호 17), 프라이머 AmpCr: 5'-GTTGGGGTAGTTGCGATTGG-3'(서열번호 18). 아울러, 양성대조군으로 프라이머 T7-p: 5'-AATACGACTCACTATAG-3'(서열번호 19)와 프라이머 SP6: 5'-GATTTAGGTGACACTATAG-3'(서열번호 20)을 작제하고, 음성대조군으로 프라이머 T7-n: 5'-AATACGACTCACTATAG-3'(서열번호 21)과 프라이머 T3: 5'-ATTAACCCTCACTAAAG-3'(서열번호 22)을 작제하였다.Each probe was constructed to include an amine group at the 5 'end using a gene resistant to beta-lactam antibiotics. Each probe was prepared by amplification by PCR using the following primers. The base sequences of the primers for amplifying the respective resistance genes are as follows: primer TEMf: 5'-TCGGGGAAATGTGCGCG-3 '(SEQ ID NO: 1), primer TEMr: 5'-TGCTTAATCAGTGAGGCACC-3' (SEQ ID NO: 2), primer SHV -1f: 5'-AAGATCCACTATCGCCAGGAGG-3 '(SEQ ID NO: 3), Primer SHV-1r: 5'-ATTCAGTTCGTTTCCCAGCGG-3' (SEQ ID NO: 4), Primer MEN-1f: 5'-TCCTCTCTTCCAGA-3 '(SEQ ID NO: 5) ), Primer MEN-1r: 5'-CAGCGCTTTTCGCGTCTAAG-3 '(SEQ ID NO: 6), primer CMY-1f: 5'-ATGCAACAACGACAATCCATC-3' (SEQ ID NO: 7), primer CMY-1r: 5'-GTTGGGTAGTTGCGATTGG-3 ' (SEQ ID NO: 8), Primer OXY-1f: 5'-CAGATCTCGAGAAGCGTTCA-3 '(SEQ ID NO: 9), Primer OXY-1r: 5'-ACCTCTTTGCGGTTTTTCGC-3' (SEQ ID NO: 10), Primer FOXf: 5'-CACCACGAGAATAACCAT- 3 '(SEQ ID NO: 11), Primer FOXr: 5'-ATGTGGACGCCTTGAACT-3' (SEQ ID NO: 12), Primer PSE-1f: 5'-AATGGCATTCAGCGCTTCCC-3 '(SEQ ID NO: 13), Primer PSE-1r: 5'- GGGGCTTGATGCTCACTC C-3 '(SEQ ID NO: 14), Imer OXA-1f: 5'-TCAACTTTCAAGATCGCA-3 '(SEQ ID NO: 15), Primer OXA-1r: 5'-GTGTGTTTAGAATGGTGA-3' (SEQ ID NO: 16), Primer AmpCf: 5'-CTACGGTCTGGCTGCTA-3 '(SEQ ID NO: 17 ), Primer AmpCr: 5'-GTTGGGGTAGTTGCGATTGG-3 '(SEQ ID NO: 18). In addition, primer T7-p: 5'-AATACGACTCACTATAG-3 '(SEQ ID NO: 19) and primer SP6: 5'-GATTTAGGTGACACTATAG-3' (SEQ ID NO: 20) were constructed as a positive control, and primer T7-n: as a negative control. 5'-AATACGACTCACTATAG-3 '(SEQ ID NO: 21) and Primer T3: 5'-ATTAACCCTCACTAAAG-3' (SEQ ID NO: 22) were constructed.
전기 작제된 탐침들을 완충용액(350mM sodium bicarbonate, pH 9.0)에 각각 1M의 농도로 용해시키고, 알데히드기가 결합된 슬라이드(silylated slide, CSS-100, CEL, Houston, TX, USA) 표면에 어레이어(Nano-Plotter, GeSiM, Germany)를이용하여 크기 150mm, 간격 300mm으로 스파팅(spotting)을 수행한 다음, 시프염기반응을 수행하였다. 이어, 0.2%(w/v) 소디움 도데실설페이트(sodium dodecyl sulfate, SDS) 용액으로 슬라이드를 세척하고, 3차 증류수로 세척한 다음, NaBH4용액(0.1g NaBH4, 30ml phosphate buffered saline(PBS), 10ml ethanol)에 5분 동안 침지하여 아민이 결합되지 않은 알데히드를 환원시킨 후, 3차 증류수로 세척하고, 건조시켜서 DNA칩을 제조하였다.Electrolyzed probes were dissolved in a buffer solution (350 mM sodium bicarbonate, pH 9.0) at a concentration of 1 M each, and an arrayer (silylated slide, CSS-100, CEL, Houston, TX, USA) was placed on the surface of the arrayer ( Nano-Plotter, GeSiM, Germany) using a spotting (size 150mm, interval 300mm) using a seed base reaction. Subsequently, the slides were washed with a 0.2% (w / v) sodium dodecyl sulfate (SDS) solution, washed with tertiary distilled water, and then washed with NaBH 4 solution (0.1 g NaBH 4 , 30 ml phosphate buffered saline (PBS). ), Immersed in 10 ml ethanol for 5 minutes to reduce the amine unbound aldehyde, washed with tertiary distilled water, and dried to prepare a DNA chip.
실시예 2: 시료의 수득 Example 2 Obtaining a Sample
베타-락탐 항생제 내성병원균에서 분리한 OXA-1, MEN-1, SHV-1, OXY-1 및 AmpC의 DNA를 주형으로 하고, 25mM dATP, 25mM dGTP, 25mMdTP, 1mM dCTP, 1mM Cy5-dCTP와 서열번호 1 내지 26의 서열을 갖는 프라이머를 모두 사용하여 PCR을 수행한 다음, PCR 산물과 양성 대조군을 5㎕씩 혼합하고, 건조시켜서 시료를 수득하였다.DNAs of OXA-1, MEN-1, SHV-1, OXY-1 and AmpC isolated from beta-lactam antibiotic resistant pathogens were used as templates, and 25mM dATP, 25mM dGTP, 25mMdTP, 1mM dCTP, 1mM Cy5-dCTP and sequence PCR was performed using all primers having the sequences 1 to 26, and then 5 μl of the PCR product and the positive control were mixed and dried to obtain a sample.
실시예 3: 보합반응(hybridization) Example 3 Hybridization
실시예 1에서 제작된 DNA 칩에 15㎕의 보합반응용액(hybridization solution: 5xSSC(8.77g/L NaCl, 4.41g/L sodium citrate, pH 7.0), 0.2% SDS, 1mM dNTP)을 가하고, 커버슬라이드를 덮은 후, 65℃에서 2시간동안전보합반응(prehybridization)을 수행하였다. 이어, 20㎕의 보합반응용액에 용해시킨 실시예 2에서 수득한 각각의 시료를 95℃에서 5분간 변성(denaturation) 시킨 후, DNA 칩에 있는 용액과 교체하고, 65℃에서 보합반응을 수행하였다. 그런 다음, 보합반응이 종결된 DNA 칩을 2xSSC, 0.1% SDS 용액으로 5분간 상온에서 4번 세척하고, 0.1xSSC, 0.1% SDS용액으로 5분간 37℃에서 2번, 65℃에서 1번 세척하였다. 그 후, DNA 을 스캐너(Scan Array 5000, GSI Lumonics Inc., U.S.A.)로 스캐닝하여 결과를 분석하였다(참조: 도 1, 도 2, 도 3, 도 4, 도 5).15 μl of a hybridization solution (5xSSC (8.77 g / L NaCl, 4.41 g / L sodium citrate, pH 7.0), 0.2% SDS, 1 mM dNTP) was added to the DNA chip prepared in Example 1, and the cover slide was added. After covering, prehybridization was performed at 65 ° C. for 2 hours. Subsequently, each sample obtained in Example 2 dissolved in 20 µl of a hybridization reaction solution was denatured at 95 ° C for 5 minutes, replaced with a solution in a DNA chip, and then subjected to a hybridization reaction at 65 ° C. . Then, the DNA chip in which the reaction was terminated was washed 4 times at room temperature for 5 minutes with 2xSSC and 0.1% SDS solution, and twice at 37 ° C and once at 65 ° C for 5 minutes with 0.1xSSC and 0.1% SDS solution. . Thereafter, the DNA was scanned with a scanner (Scan Array 5000, GSI Lumonics Inc., U.S.A.) to analyze the results (see FIGS. 1, 2, 3, 4, and 5).
도 1은 본 발명의 DNA 칩을 사용하여 OXA-1의 DNA를 검출한 결과를 나타내는 사진이고; 도 2는 본 발명의 DNA 칩을 사용하여 MEN-1의 DNA를 검출한 결과를 나타내는 사진이며; 도 3은 본 발명의 DNA 칩을 사용하여 SHV-1의 DNA를 검출한 결과를 나타내는 사진이고; 도 4는 본 발명의 DNA 칩을 사용하여 OXY-1의 DNA를 검출한 결과를 나타내는 사진이며; 및, 도 5는 본 발명의 DNA 칩을 사용하여 AmpC의 DNA를 검출한 결과를 나타내는 사진이다. 전기 도 1 내지 도 5에서, 1번에는 PSE-1이 위치하고, 2번에는 OXA-1이 위치하며, 3번에는 FOX가 위치하고, 4번에는 MEN-1이 위치하며, 5번에는 CMY-1이 위치하고, 6번에는 TEM이 위치하며, 7번에는 SHV-1이 위치하고, 8번에는 OXY-1이 위치하며, 9번에는 AmpC가 위치하고, negative에는 인간의 종양괴사인자 수용체 결합인자(human TNF receptor associated factor)가 위치하며, positive에는 인간의 TRAIL 수용체(human TRAIL receptor 2)가 위치한다. 도 1 내지 도 5에서 보듯이, 베타-락타메이즈인 OXA-1, MEN-1, SHV-1, OXY-1 및 AmpC의 DNA는 각각 DNA 칩의 탐침과 반응할 수 있는 위치에서만 정확히 검출됨을알 수 있었다.1 is a photograph showing the results of detecting DNA of OXA-1 using the DNA chip of the present invention; 2 is a photograph showing the results of detecting the DNA of MEN-1 using the DNA chip of the present invention; 3 is a photograph showing the results of detecting the DNA of SHV-1 using the DNA chip of the present invention; 4 is a photograph showing a result of detecting DNA of OXY-1 using the DNA chip of the present invention; And FIG. 5 is a photograph showing the results of detecting AmpC DNA using the DNA chip of the present invention. 1 to 5, PSE-1 is located at 1, OXA-1 is located at 2, FOX is located at 3, MEN-1 is located at 4, CMY-1 is located at 5 Is located, TEM is located at 6, SHV-1 is located at 7, OXY-1 is located at 8, AmpC is located at 9, human tumor necrosis factor receptor binding factor (human TNF) at negative The receptor associated factor is located, and the positive TRAIL receptor (human TRAIL receptor 2) is located. As shown in FIGS. 1 to 5, the beta-lactamase DNAs of OXA-1, MEN-1, SHV-1, OXY-1 and AmpC are detected only at positions capable of reacting with the DNA chip probes, respectively. Could.
이상에서 상세히 설명하고 입증하였듯이, 본 발명은 베타-락탐계 항생물질에 대한 내성을 가지는 균주를 선별하기 위한 유전형 분석키트 및 전기 분석키트를 사용하여 베타-락탐계 항생물질에 대한 내성을 가지는 균주를 선별하는 방법을 제공한다. 본 발명의 베타-락탐계 항생제 내성균을 선별하기 위한 유전형 분석키트는 베타-락타메이즈 유전자와 상보적으로 결합할 수 있는 탐침을 포함하는 DNA칩, 시료로부터 채취한 DNA를 PCR방법으로 증폭시키기 위한 프라이머 및 전기 DNA칩과 보합반응되는 증폭된 DNA를 탐지하기 위한 표지수단인 Cy5-dCTP를 포함하고, 전기 분석키트를 이용하여 베타-락탐계 항생제 내성균을 선별하는 방법은 유전형 분석키트의 프라이머를 사용하여 검사할 시료로부터 채취한 DNA를 증폭시키는 단계; 전기 증폭된 DNA를 DNA칩에 적용하여, 증폭된 DNA와 탐침을 보합반응시키는 단계; 및, 탐침과 보합반응된 DNA를 검출하는 단계를 포함한다. 본 발명의 유전형 분석키트를 이용하면, 간단한 방법으로 항생제에 내성을 가지는 병원성 균주를 탐지할 수 있으므로, 병원성 세균의 연구에 널리 활용될 수 있을 것이다.As described and demonstrated in detail above, the present invention uses a genotyping kit and an electroanalytical kit for screening strains resistant to beta-lactam antibiotics to identify strains resistant to beta-lactam antibiotics. Provide a method for screening. The genotyping kit for selecting beta-lactam antibiotic resistant bacteria of the present invention is a DNA chip comprising a probe capable of complementarily binding to a beta-lactamase gene, and a primer for amplifying DNA obtained from a sample by PCR. And Cy5-dCTP, which is a labeling means for detecting amplified DNA that reacts with the electric DNA chip. The method for selecting beta-lactam antibiotic resistance bacteria using an electric analysis kit is performed by using a primer of a genotyping assay kit. Amplifying the DNA collected from the sample to be tested; Applying the electrically amplified DNA to the DNA chip to perform a hybrid reaction between the amplified DNA and the probe; And detecting DNA that has been reacted with the probe. By using the genotyping kit of the present invention, a pathogenic strain resistant to antibiotics can be detected by a simple method, and thus, it can be widely used for the study of pathogenic bacteria.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시태양일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail the specific parts of the present invention, for those skilled in the art, these specific descriptions are only preferred embodiments, which are not intended to limit the scope of the present invention. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
<110> DISGENE <120> Genotyping kit for Selecting Antibiotics Resistant Bacteria <130> DP01204 <160> 22 <170> KopatentIn 1.6 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer TEMf <400> 1 tcggggaaat gtgcgcg 17 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer TEMr <400> 2 tgcttaatca gtgaggcacc 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer SHV-1f <400> 3 aagatccact atcgccagga gg 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer SHV-1r <400> 4 attcagttcg tttcccagcg g 21 <210> 5 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> primer MEN-1f <400> 5 tcctctcttc caga 14 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer MEN-1r <400> 6 cagcgctttt cgcgtctaag 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer CMY-1f <400> 7 atgcaacaac gacaatccat c 21 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer CMY-1r <400> 8 gttgggtagt tgcgattgg 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OXY-1f <400> 9 cagatctcga gaagcgttca 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer OXY-1r <400> 10 acctctttgc ggtttttcgc 20 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer FOXf <400> 11 caccacgaga ataaccat 18 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer FOXr <400> 12 atgtggacgc cttgaact 18 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer PSE-1f <400> 13 aatggcattc agcgcttccc 20 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer PSE-1r <400> 14 ggggcttgat gctcactcc 19 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer OXA-1f <400> 15 tcaactttca agatcgca 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer OXA-1r <400> 16 gtgtgtttag aatggtga 18 <210> 17 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer AmpCf <400> 17 ctacggtctg gctgcta 17 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer AmpCr <400> 18 gttggggtag ttgcgattgg 20 <210> 19 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer T7-p <400> 19 aatacgactc actatag 17 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer SP6 <400> 20 gatttaggtg acactatag 19 <210> 21 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer T7-n <400> 21 aatacgactc actatag 17 <210> 22 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer T3 <400> 22 attaaccctc actaaag 17<110> DISGENE <120> Genotyping kit for Selecting Antibiotics Resistant Bacteria <130> DP01204 <160> 22 <170> KopatentIn 1.6 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer TEMf <400> 1 tcggggaaat gtgcgcg 17 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer TEMr <400> 2 tgcttaatca gtgaggcacc 20 <210> 3 <211> 22 < 212> DNA <213> Artificial Sequence <220> <223> primer SHV-1f <400> 3 aagatccact atcgccagga gg 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer SHV-1r <400> 4 attcagttcg tttcccagcg g 21 <210> 5 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> primer MEN-1f <400> 5 tcctctcttc caga 14 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer MEN-1r <400> 6 cagcgctttt cgcgtctaag 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer CMY-1f <400> 7 atgcaacaac gacaatccat c 21 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer CMY-1r <400> 8 gttgggtagt tgcgattgg 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OXY-1f <400> 9 cagatctcga gaagcgttca 20 <210> 10 <211> 20 <212> DNA < 213> Artificial Sequence <220> <223> primer OXY-1r <400> 10 acctctttgc ggtttttcgc 20 <210> 11 <211> 18 <212> DNA <213> Arti ficial Sequence <220> <223> primer FOXf <400> 11 caccacgaga ataaccat 18 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer FOXr <400> 12 atgtggacgc cttgaact 18 < 210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer PSE-1f <400> 13 aatggcattc agcgcttccc 20 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer PSE-1r <400> 14 ggggcttgat gctcactcc 19 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer OXA-1f <400> 15 tcaactttca agatcgca 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer OXA-1r <400> 16 gtgtgtttag aatggtga 18 <210> 17 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer AmpCf <400> 17 ctacggtctg gctgcta 17 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer AmpCr <400> 18 gttggggtag ttgcgattgg 20 <210> 19 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer T7-p <400> 19 aatacgactc actatag 17 < 210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer SP6 <400> 20 gatttaggtg acactatag 19 <210> 21 <211> 17 <212> DNA <213> Artificial Sequence <220 > <223> primer T7-n <400> 21 aatacgactc actatag 17 <210> 22 <211> 17 <212> DNA <2 13> Artificial Sequence <220> <223> primer T3 <400> 22 attaaccctc actaaag 17
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| WO1992019763A1 (en) * | 1991-05-06 | 1992-11-12 | Baxter Diagnostics Inc. | Rapid inducible beta-lactamase screen test |
| US5571693A (en) * | 1990-07-16 | 1996-11-05 | American Cyanamid Company | DNA sequences and amino acid sequences of class B beta lactamase enzymes from bacteroides fragilis |
| JPH11169196A (en) * | 1997-11-11 | 1999-06-29 | Becton Dickinson & Co | Detection of vancomycin-resistant bacterilim and culture medium for detection |
| WO2000071702A1 (en) * | 1999-05-25 | 2000-11-30 | Panorama Research, Inc. | Interaction-activated proteins |
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| US5571693A (en) * | 1990-07-16 | 1996-11-05 | American Cyanamid Company | DNA sequences and amino acid sequences of class B beta lactamase enzymes from bacteroides fragilis |
| WO1992019763A1 (en) * | 1991-05-06 | 1992-11-12 | Baxter Diagnostics Inc. | Rapid inducible beta-lactamase screen test |
| JPH11169196A (en) * | 1997-11-11 | 1999-06-29 | Becton Dickinson & Co | Detection of vancomycin-resistant bacterilim and culture medium for detection |
| WO2000071702A1 (en) * | 1999-05-25 | 2000-11-30 | Panorama Research, Inc. | Interaction-activated proteins |
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