KR20090124020A - Oligonucleotide probe set for dectecting food-borne pathogens and microarray containing the probe set - Google Patents

Oligonucleotide probe set for dectecting food-borne pathogens and microarray containing the probe set Download PDF

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KR20090124020A
KR20090124020A KR1020080050010A KR20080050010A KR20090124020A KR 20090124020 A KR20090124020 A KR 20090124020A KR 1020080050010 A KR1020080050010 A KR 1020080050010A KR 20080050010 A KR20080050010 A KR 20080050010A KR 20090124020 A KR20090124020 A KR 20090124020A
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김해영
김현중
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Abstract

PURPOSE: An oligonecleotide probe set for detecting bacteria causing food poisoning is provided to quickly and accurately detect and identify bacteria. CONSTITUTION: An oligonucleotide probe set for detecting bacteria causing food poisoning contains an oligonucleotide probe having one oligonucleotide among sequence numbers 31 to 40 for detecting Clostridium botulinum, an oligonucleotide probe having one oligonucleotide among sequence numbers 41 to 50 for detecting Clostridium perfringens, an oligonucleotide probe having one oligonuelcotide among sequence numbers 51 to 60 for detecting Campylobacter jejuni, or an oligonucleotide probe having one oligonucleotide among sequence numbers 61 to 70 for detecting Vibrio parahaemolyticus.

Description

식중독균 검출용 올리고뉴클레오티드 프로브 세트 및 상기 프로브 세트를 포함하는 마이크로어레이{Oligonucleotide probe set for dectecting food-borne pathogens and microarray containing the probe set}Oligonucleotide probe set for dectecting food-borne pathogens and microarray containing the probe set}

본 발명은 식중독균 검출용 올리고뉴클레오티드 프로브 세트, 상기 프로브 세트를 포함하는 마이크로어레이 및 키트에 관한 것이다.The present invention relates to an oligonucleotide probe set for detecting food poisoning bacteria, a microarray and a kit including the probe set.

식중독균은 세계적으로 인간의 건강에 주요한 문제점을 일으키고 있으며 식품산업, 공공 건강 증진, 미생물 테러 등의 분야에서 식중독균의 빠른 검출이 필요한 실정이다. 인간에 질병을 일으키는 균들 중 주요한 식중독균들은 다음과 같다: 바실러스 세레우스(Bacillus cereus), 리스테리아 모노시토게네스(Listeria monocytogenes), 스태필로코커스 오레우스(Staphylococcus aureus), 클로스트리디움 보툴리눔(Clostridium botulinum), 클로스트리디움 퍼프린젠스(Clostridium perfringens), 캄필로박터 제주니(Campylobacter jejuni), 비브리오 파라해모리티쿠스(Vibrio parahaemolyticus), 여시니아 엔테로콜리티카(Yersinia enterocolitica), 에세리키아 콜라이(Escherichia coli) O157:H7 및 살모넬라 엔테리카 세로바 티피무리움(Salmonella enterica serovar Typhimurium). 이러한 식중 독균의 검출은 인간의 건강과 식품 산업에서 매우 중요시되고 있으며, 식중독 검출에 있어서 민감도 (sensitivity), 검출 시간 (rapidity) 및 특이적인 검출(specificity)이 가장 중요한 요건이다. 기존에 알려져 있는 생화학적인 식중독균의 진단 방법은 일반적으로 2-3일 정도의 시간이 필요하며 한 번의 실험으로 다중의 병원균의 검출에는 무리가 있었다.Food poisoning bacteria are causing major problems in human health around the world, and there is a need for rapid detection of food poisoning bacteria in the fields of food industry, public health promotion and microbial terrorism. The major food poisoning bacteria that cause disease in humans are: Bacillus cereus ), Listeria monocytogenes , Staphylococcus aureus), Clostridium botulinum (Clostridium botulinum ), Clostridium perfringens , Campylobacter jejuni ), Vibrio parahaemoritis ( Vibrio) parahaemolyticus ), Yersinia enterocolitica , Escherichia coli ) O157: H7 and Salmonella enterica serova typhimurium enterica serovar Typhimurium). Detection of such food poisoning bacteria is very important in human health and food industry, and sensitivity, detection time and specificity are the most important requirements in detecting food poisoning. Known biochemical methods of food poisoning bacteria generally require 2-3 days, and it is difficult to detect multiple pathogens in one experiment.

몇몇의 DNA를 이용한 식중독균의 검출방법이 보고되었으나, 이 방법들 중 PCR (polymerase chain reaction, 중합효소연쇄반응)은 가장 일반적으로 사용된 방법이다. 그러나 PCR은 사용하는 프라이머의 숫자에 제한이 있으므로 다중의 식중독균의 검출에 문제점을 가지고 있다. 또한 비슷한 속 균주들 사이에서 DNA 서열의 유사성이나 다양성에 의한 위양성(false-positive)이나 위음성(false-negative)을 나타낼 수 있는 가능성이 있다. DNA 서열에 중심을 둔 마이크로어레이 실험 방법은 유전자 발현, 지노타이핑(genotyping) 및 단일염기다형성(single-nucleotide polymorphism) 등의 실험에 사용되어 왔다(Chizhikov et al. 2002. J. Clin. Microbiol. 40: 2398-2407). 또한 마이크로어레이 실험방법은 한 번의 실험으로 다수의 식중독균을 검출할 수 있는 잠재능력 때문에 새로운 식중독균 검출 방법으로 제안되었다 (Al-khaldi et al. 2002. J. AOAC Int. 85: 906-910).Although some methods of detecting food poisoning bacteria using DNA have been reported, PCR (polymerase chain reaction) is the most commonly used method. However, PCR has a problem in detecting multiple food poisoning bacteria because of the limited number of primers used. In addition, there is a possibility of showing false-positive or false-negative by similarity or diversity of DNA sequences among similar genus strains. Microarray experiments centered on DNA sequences have been used for experiments such as gene expression, genotyping and single-nucleotide polymorphism (Chizhikov et al. 2002. J. Clin. Microbiol. 40). : 2398-2407). In addition, the microarray test method has been proposed as a new method for detecting food poisoning bacteria because of the potential for detecting a plurality of food poisoning bacteria in one experiment (Al-khaldi et al. 2002. J. AOAC Int. 85: 906-910).

최근 들어, cDNA 및 올리고뉴클레오티드 마이크로어레이 기술이 병원성 미생물의 분석에 적용되어 왔으며 (Burton et al. 2005. Molecular and Cellular Probes. 19: 349-357), 하나의 칩으로 4종의 식중독균을 검출할 수 있는 연구가 보고되었다 (Sergeev et al. 2004. Biosensors and Bioelectronics. 20: 684-698). 이러한 기존의 연구들에서 제작된 프로브들은 미생물의 flagelline 유전자나 독성 유전자 (병원성 관련 유전자 포함), 16S rRNA 또는 23S rRNA 서열에서 제작되었다. 그러나 이러한 표적 유전자들은 같은 속의 미생물들 간에 그 서열을 공유하고 있으며, 이는 cross-hybridization의 결과를 초래할 가능성이 높다. 또한, 이러한 연구들에서 혼성화 전에 PCR을 수행하는데, 이 방법은 사용 가능한 프로브의 수가 제한적일 수밖에 없으며 이는 다수의 병원균을 검출하는데 무리가 있다 (Chizhikov et al. 2001. Appl. Environ. Microbiol. 67: 3258-3263).Recently, cDNA and oligonucleotide microarray techniques have been applied to the analysis of pathogenic microorganisms (Burton et al. 2005. Molecular and Cellular Probes. 19: 349-357), and one chip can detect four food poisoning bacteria. Studies have been reported (Sergeev et al. 2004. Biosensors and Bioelectronics. 20: 684-698). Probes produced in these previous studies were constructed from microbial flagelline genes or virulence genes (including pathogenic genes), 16S rRNA or 23S rRNA sequences. However, these target genes share their sequence among microorganisms of the same genus, which is likely to result in cross-hybridization. In addition, in these studies PCR is performed prior to hybridization, which is limited to the number of available probes, which makes it difficult to detect a large number of pathogens (Chizhikov et al. 2001. Appl. Environ. Microbiol. 67: 3258). -3263).

2006년 1월을 기준으로 약 270개 이상의 미생물에 대한 게놈 서열이 National Center for Biotechnology information (NCBI, http://www.ncbi.nih.gov/genomes/lproks.cgi)에 공개되어 있으며, 대부분의 식중독균에 대한 게놈 염기서열 분석 작업이 끝났거나 진행 중에 있다.As of January 2006, genomic sequences for more than 270 microorganisms have been published in the National Center for Biotechnology information (NCBI, http://www.ncbi.nih.gov/genomes/lproks.cgi ). Genomic sequencing work for food poisoning bacteria is finished or in progress.

한국특허등록 제10-611056호에는 병원성 미생물 검출을 위한 올리고뉴클레오티드마이크로칩 및 이를 이용한 병원성 미생물 검출방법이 개시되어 있으며, 한국특허등록 제10-671501호에는 식중독 검출용 프라이머 및 이를 이용한 세균성 식중독 검출방법이 개시되어 있으나, 본 발명의 프로브 서열과는 상이하다.Korean Patent Registration No. 10-611056 discloses an oligonucleotide microchip for detecting pathogenic microorganisms and a pathogenic microorganism detection method using the same. Korean Patent Registration No. 10-671501 discloses a food poisoning detection primer and a bacterial food poisoning detection method using the same. Is disclosed, but differs from the probe sequences of the present invention.

본 발명은 상기와 같은 요구에 의해 안출된 것으로서, 본 발명에서는 각각의 식중독균의 게놈 서열로부터 각각의 식중독균에 특이적인 70-mer 올리고뉴클레오티드 프로브들을 선별하였으며, 선별된 프로브들을 이용하여 "FBP-1"이라고 명명한 식중독균 검출용 마이크로어레이 칩을 제작하였다. 이 제작한 칩을 이용하여 다양한 식중독균 및 비병원성 균들의 게놈 DNA와 반응시켜 그 특이성을 증명함으로써, 본 발명을 완성하였다.The present invention has been made in accordance with the above requirements, and in the present invention, 70-mer oligonucleotide probes specific for each food poisoning bacterium were selected from the genome sequence of each food poisoning bacterium, and the "FBP-1" was selected using the selected probes. A microarray chip for detecting food poisoning bacteria was named. The produced chip was used to react with genomic DNA of various food poisoning bacteria and non-pathogenic bacteria to prove its specificity, thereby completing the present invention.

상기 과제를 해결하기 위해, 본 발명은 신속하고 정확한 검출이 가능한 식중독균 검출용 올리고뉴클레오티드 프로브 세트를 제공한다.In order to solve the above problems, the present invention provides an oligonucleotide probe set for detecting food poisoning bacteria capable of rapid and accurate detection.

또한, 본 발명은 상기 올리고뉴클레오티드 프로브 세트를 포함하는 마이크로어레이를 제공한다.The present invention also provides a microarray comprising the oligonucleotide probe set.

또한, 본 발명은 상기 올리고뉴클레오티드 프로브 세트를 이용하여 식중독균을 검출하는 방법을 제공한다.The present invention also provides a method for detecting food poisoning bacteria using the oligonucleotide probe set.

또한, 본 발명은 상기 올리고뉴클레오티드 프로브 세트를 포함하는 식중독균 검출 키트를 제공한다.In addition, the present invention provides a food poisoning bacteria detection kit comprising the oligonucleotide probe set.

본 발명에 따르면, 본 발명의 프로브는 식품 산업에서 문제가 되고 있는 식중독균에 대한 빠른 검출 및 동정에 활용될 수 있을 것이라 사료되며, 또한 게놈 서열 비교 방법 및 DNA 칩을 이용한 마이크로어레이 실험 방법이 미생물 병원균에 대한 안전성 연구에 적용될 수 있는 가능성을 보여주었다. 또한, 비교 유전체학의 미생물 분류 연구에 대한 새로운 적용방법을 제시하였다.According to the present invention, the probe of the present invention may be utilized for the rapid detection and identification of food poisoning bacteria, which is a problem in the food industry, and the method of comparing genomic sequences and microarray experiments using DNA chips is a microbial pathogen. It has been shown to be applicable to safety studies. In addition, a new application method for microbial classification studies in comparative genomics is presented.

본 발명의 목적을 달성하기 위하여, 본 발명은In order to achieve the object of the present invention, the present invention

서열번호 31 내지 40의 올리고뉴클레오티드로 이루어진 군으로부터 선택되는 하나 이상의 올리고뉴클레오티드를 포함하는, 클로스트리디움 보툴리눔(Clostridium botulinum) 검출용 올리고뉴클레오티드 프로브;Oligonucleotide of SEQ ID NO: 31 to 40 one oligonucleotide selected from the group consisting of a nucleotide or more, including nucleotide, Clostridium botulinum (Clostridium botulinum ) oligonucleotide probe for detection;

서열번호 41 내지 50의 올리고뉴클레오티드로 이루어진 군으로부터 선택되는 하나 이상의 올리고뉴클레오티드를 포함하는, 클로스트리디움 퍼프린젠스(Clostridium perfringens) 검출용 올리고뉴클레오티드 프로브;Oligonucleotide of SEQ ID NO: 41 to 50 one oligonucleotide selected from the group consisting of a nucleotide or more, including nucleotide, Clostridium perfringens (Clostridium perfringens ) detection oligonucleotide probes;

서열번호 51 내지 60의 올리고뉴클레오티드로 이루어진 군으로부터 선택되는 하나 이상의 올리고뉴클레오티드를 포함하는, 캄필로박터 제주니(Campylobacter jejuni) 검출용 올리고뉴클레오티드 프로브; 및Campylobacter, comprising one or more oligonucleotides selected from the group consisting of oligonucleotides of SEQ ID NOs: 51-60 jejuni ) detection oligonucleotide probes; And

서열번호 61 내지 70의 올리고뉴클레오티드로 이루어진 군으로부터 선택되는 하나 이상의 올리고뉴클레오티드를 포함하는, 비브리오 파라해모리티쿠스(Vibrio parahaemolyticus) 검출용 올리고뉴클레오티드 프로브로 이루어진 군으로부터 선택된 하나 이상의 프로브를 포함하는 식중독균 검출용 올리고뉴클레오티드 프로브 세트를 제공한다.Vibrio parahaemoriticus, comprising one or more oligonucleotides selected from the group consisting of oligonucleotides of SEQ ID NOs: 61-70 parahaemolyticus ) provides a set of oligonucleotide probes for detecting food poisoning bacteria comprising one or more probes selected from the group consisting of oligonucleotide probes for detection.

본 발명의 일 구현예에 따른 식중독균 검출용 올리고뉴클레오티드 프로브 세 트에서, 클로스트리디움 보툴리눔(Clostridium botulinum) 검출용 올리고뉴클레오티드 프로브는 서열번호 31 내지 40의 올리고뉴클레오티드로 이루어진 군으로부터 선택되는 하나 이상의 올리고뉴클레오티드를 포함할 수 있으며, 바람직하게는 서열번호 31 내지 40의 올리고뉴클레오티드 모두를 포함할 수 있다.The three oligonucleotides for sikjungdokgyun detection according to one embodiment of the invention a nucleotide probe agent, Clostridium botulinum (Clostridium botulinum ) oligonucleotide probe for detection may include one or more oligonucleotides selected from the group consisting of oligonucleotides of SEQ ID NOs: 31 to 40, and preferably include all of the oligonucleotides of SEQ ID NOs: 31 to 40.

본 발명의 일 구현예에 따른 식중독균 검출용 올리고뉴클레오티드 프로브 세트에서, 클로스트리디움 퍼프린젠스(Clostridium perfringens) 검출용 올리고뉴클레오티드 프로브는 서열번호 41 내지 50의 올리고뉴클레오티드로 이루어진 군으로부터 선택되는 하나 이상의 올리고뉴클레오티드를 포함할 수 있으며, 바람직하게는 서열번호 41 내지 50의 올리고뉴클레오티드 모두를 포함할 수 있다.In the nucleotide probe set up for sikjungdokgyun detection according to one embodiment of the present invention, Clostridium perfringens (Clostridium perfringens ) oligonucleotide probe for detection may include one or more oligonucleotides selected from the group consisting of oligonucleotides of SEQ ID NOs: 41 to 50, and preferably include all of the oligonucleotides of SEQ ID NOs: 41 to 50.

본 발명의 일 구현예에 따른 식중독균 검출용 올리고뉴클레오티드 프로브 세트에서, 캄필로박터 제주니(Campylobacter jejuni) 검출용 올리고뉴클레오티드 프로브는 서열번호 51 내지 60의 올리고뉴클레오티드로 이루어진 군으로부터 선택되는 하나 이상의 올리고뉴클레오티드를 포함할 수 있으며, 바람직하게는 서열번호 51 내지 60의 올리고뉴클레오티드 모두를 포함할 수 있다.In the oligonucleotide probe set for detecting food poisoning bacteria according to an embodiment of the present invention, Campylobacter jejuni jejuni ) The oligonucleotide probe for detection may include one or more oligonucleotides selected from the group consisting of oligonucleotides of SEQ ID NOs: 51 to 60, and preferably may include all of the oligonucleotides of SEQ ID NOs: 51 to 60.

본 발명의 일 구현예에 따른 식중독균 검출용 올리고뉴클레오티드 프로브 세트에서, 비브리오 파라해모리티쿠스(Vibrio parahaemolyticus) 검출용 올리고뉴클레오티드 프로브는 서열번호 61 내지 70의 올리고뉴클레오티드로 이루어진 군으로부터 선택되는 하나 이상의 올리고뉴클레오티드를 포함할 수 있으며, 바람직하게는 서열번호 61 내지 70의 올리고뉴클레오티드 모두를 포함할 수 있다.In the oligonucleotide probe set for detecting food poisoning bacteria according to an embodiment of the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus ) oligonucleotide probe for detection may include one or more oligonucleotides selected from the group consisting of oligonucleotides of SEQ ID NOs: 61 to 70, and preferably may include all of the oligonucleotides of SEQ ID NOs: 61 to 70.

본 발명의 일 구현예에 따른 식중독균 검출용 올리고뉴클레오티드 프로브 세 트는 가장 바람직하게는 서열번호 31 내지 70의 올리고뉴클레오티드 모두를 포함할 수 있다.Oligonucleotide probe set for detecting food poisoning bacteria according to an embodiment of the present invention may most preferably include all of the oligonucleotides of SEQ ID NOs: 31 to 70.

본 명세서에 있어서, "프로브"란 상보적인 단일가닥 표적 서열과 혼성화하여 이중가닥 분자 (혼성체)를 형성하는 단일가닥 핵산 서열을 말한다.As used herein, "probe" refers to a single stranded nucleic acid sequence that hybridizes with a complementary single stranded target sequence to form a double stranded molecule (hybrid).

본 명세서에 있어서, 프로브로서 이용된 올리고뉴클레오티드는 또한 뉴클레오티드 유사체(analogue), 예를 들면, 포스포로티오에이트(phosphorothioate), 알킬포스포로티오에이트 또는 펩티드 핵산(peptide nucleic acid)를 포함할 수 있거나 또는 삽입 물질(intercalating agent)를 포함할 수 있다.As used herein, oligonucleotides used as probes may also include nucleotide analogs, such as phosphorothioate, alkylphosphothioate or peptide nucleic acid, or It may comprise an intercalating agent.

본 발명은 또한, 본 발명의 일 실시예에 따른 하나 이상의 올리고뉴클레오티드 프로브 세트가 고정화되어 있는 기판을 갖는 마이크로어레이를 제공한다.The invention also provides a microarray having a substrate on which at least one oligonucleotide probe set is immobilized according to one embodiment of the invention.

본 발명의 마이크로어레이에 있어서, "마이크로어레이"란 기판 상에 올리고뉴클레오티드의 그룹이 높은 밀도로 고정화되어 있는 것으로서, 상기 올리고뉴클레오티드 그룹은 각각 일정한 영역에 고정화되어 있는 마이크로어레이를 의미한다. 이러한 마이크로어레이는 당업계에 잘 알려져 있다. 마이크로어레이에 관하여는 예를 들면, 미국특허 제5,445,934호 및 제5,744,305호에 개시되어 있으며, 이들 특허의 내용은 참조에 의하여 본 명세서에 포함된다. 상기 올리고뉴클레오티드 프로브 세트에 대하여는 상기 기재한 바와 같다.In the microarray of the present invention, "microarray" refers to a microarray in which a group of oligonucleotides is immobilized at a high density on a substrate, and each of the oligonucleotide groups is immobilized in a predetermined region. Such microarrays are well known in the art. Microarrays are disclosed, for example, in US Pat. Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference. The oligonucleotide probe set is as described above.

용어 "기판"은 혼성화 특성을 보유하고, 혼성화의 배경 수준이 낮게 유지되는 조건하에 올리고뉴클레오티드 프로브가 부착될 수 있는 임의의 기판을 말한다. 통상적으로, 상기 기판은 미세역가(microtiter) 플레이트, 막(예를 들면, 나일론 또는 니트로셀룰로오스) 또는 미세구(비드) 또는 칩일 수 있다. 막에 적용 또는 고정 전에, 핵산 프로브를 변형시켜 고정화를 촉진시키거나 혼성화 효율을 개선시킬 수 있다. 상기 변형은 단독중합체 테일링(homopolymer tailing), 지방족기, NH2 기, SH 기 및 카르복실기와 같은 상이한 반응성 작용기와의 커플링, 또는 바이오틴, 합텐 또는 단백질과의 커플링을 포함할 수 있다.The term “substrate” refers to any substrate to which an oligonucleotide probe can be attached under conditions that retain hybridization properties and keep the background level of hybridization low. Typically, the substrate may be a microtiter plate, membrane (eg, nylon or nitrocellulose) or microspheres (beads) or chips. Prior to application or immobilization on the membrane, nucleic acid probes can be modified to promote immobilization or to improve hybridization efficiency. Such modifications may include homopolymer tailing, coupling with different reactive functional groups such as aliphatic groups, NH 2 groups, SH groups and carboxyl groups, or coupling with biotin, hapten or protein.

본 발명은 또한, The present invention also provides

시료의 게놈 DNA를 본 발명의 일 실시예에 따른 하나 이상의 올리고뉴클레오티드 프로브 세트와 접촉시켜 상기 시료 중의 표적 서열과 프로브 서열을 혼성화시키는 단계; 및Contacting genomic DNA of a sample with one or more oligonucleotide probe sets according to one embodiment of the present invention to hybridize a target sequence and a probe sequence in the sample; And

상기 프로브와 시료 중의 표적 서열 사이의 혼성화 정도를 검출하는 단계를 포함하는 식중독균을 검출하는 방법을 제공한다.It provides a method for detecting food poisoning bacteria comprising the step of detecting the degree of hybridization between the probe and the target sequence in the sample.

상기 식중독균은 식중독을 유발하는 임의의 균일 수 있으나, 바람직하게는 클로스트리디움 보툴리눔(Clostridium botulinum), 클로스트리디움 퍼프린젠스(Clostridium perfringens), 캄필로박터 제주니(Campylobacter jejuni) 및 비브리오 파라해모리티쿠스(Vibrio parahaemolyticus)이다.The sikjungdokgyun is any uniformity of causing food poisoning. However, preferably Clostridium botulinum (Clostridium botulinum ), Clostridium perfringens ), Campylobacter jejuni ) and Vibrio parahaemolyticus .

본 명세서에 있어서, "혼성화"란 핵산의 2개의 상보적 가닥이 조합하여 이중가닥 분자 (혼성체)를 형성하는 과정을 말한다.As used herein, "hybridization" refers to the process by which two complementary strands of nucleic acid combine to form a double stranded molecule (hybrid).

본 발명의 일 구체예는, 본 발명의 방법에 있어서 상기 혼성화는 높은 엄격도 혼성화 조건하에서 수행될 수 있다. 본 구체예에서, 상기 높은 엄격도 혼성화 조건은 바람직하게는 65℃에서 동일한 몰의 Na2HPO4 및 NaH2PO4, 1mM EDTA 및 0.02% 소듐 도데실 설페이트를 포함하는 0.12 M 포스페이트 버퍼를 포함하는 것이다.In one embodiment of the invention, the hybridization in the process of the invention can be carried out under high stringency hybridization conditions. In this embodiment, the high stringency hybridization conditions preferably comprise 0.12 M phosphate buffer comprising the same moles of Na 2 HPO 4 and NaH 2 PO 4 , 1 mM EDTA and 0.02% sodium dodecyl sulfate at 65 ° C. will be.

또한, 본 명세서에 있어서, "엄격도 (stringency)"란 혼성화 및 그 후의 처리 단계 동안 존재하는 온도 및 용매 조성을 기술하기 위하여 사용되는 용어이다. 높은 엄격도 조건하에서는 고도로 상동적인 핵산 혼성체가 형성될 것이다. 즉, 충분한 정도의 상보성이 없는 혼성체는 형성되지 않을 것이다. 따라서 분석 조건의 엄격도는 혼성체를 형성하는 2 핵산 가닥 사이에 필요한 상보성의 양을 결정한다. 엄격도는 표적과 비표적 핵산과 형성된 혼성체 사이의 안정성의 차이를 최대화하기 위하여 선택된다.In addition, in this specification, "stringency" is a term used to describe the temperature and solvent composition present during hybridization and subsequent processing steps. Under high stringency conditions highly homologous nucleic acid hybrids will form. That is, hybrids without sufficient degree of complementarity will not be formed. The stringency of the assay conditions thus determines the amount of complementarity required between the two nucleic acid strands forming the hybrid. Stringency is chosen to maximize the difference in stability between the target and non-target nucleic acids and the hybrids formed.

상기 시료는 식중독균이 의심되는 미생물을 포함하는 임의의 시료일 수 있으며, 상기 시료에서 게놈 DNA를 분리하는 방법은 당업계에 통상적으로 이용되는 방법을 이용할 수 있으며, 예를 들면, DNeasy Tissue Kit (QIAGEN, Hilden, Germany)을 이용하여 게놈 DNA를 추출할 수 있다.The sample may be any sample including a microorganism suspected of food poisoning bacteria, and the method for separating genomic DNA from the sample may use a method commonly used in the art, for example, DNeasy Tissue Kit (QIAGEN , Hilden, Germany) can be used to extract genomic DNA.

본 발명의 방법에 있어서, 상기 표적 서열은 검출 가능한 표지 물질로 표지될 수 있다. 일 구현예에서, 상기 표지 물질은 형광, 인광 또는 방사성을 발하는 물질일 수 있으나, 이에 제한되지 않는다. 바람직하게는, 상기 표지 물질은 Cy-5 또는 Cy-3이다. 표적 서열의 증폭시 프라이머의 5'-말단에 Cy-5 또는 Cy-3를 표지하여 Klenow fragment를 이용하여 핵산을 합성하거나 PCR을 수행하면 표적 서열이 검출 가능한 형광 표지 물질로 표지될 수 있다. 또한, 방사성 물질을 이용한 표지 는 핵산 합성 또는 PCR 수행시 32P 또는 35S 등과 같은 방사성 동위원소를 핵산 합성 또는 PCR 반응액에 첨가하면 증폭 산물이 합성되면서 방사성이 증폭 산물에 혼입되어 증폭 산물이 방사성으로 표지될 수 있다.In the method of the present invention, the target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be, but is not limited to, a material that emits fluorescence, phosphorescence, or radiation. Preferably, the labeling substance is Cy-5 or Cy-3. When amplifying the target sequence, the 5'-terminus of the primer is labeled with Cy-5 or Cy-3 to synthesize a nucleic acid using a Klenow fragment or perform PCR to label the target sequence with a detectable fluorescent label. In addition, the label using the radioactive material may be added to the nucleic acid synthesis or PCR reaction solution by adding radioactive isotopes such as 32 P or 35 S to the nucleic acid synthesis or PCR reaction solution. Can be labeled.

본 발명의 방법에 있어서, 상기 프로브 세트는 마이크로어레이의 기판 상에 고정화될 수 있다. 마이크로어레이 기판 상에 고정화되는 올리고뉴클레오티드 프로브 세트에 대하여는 상기 기재한 바와 같다.In the method of the invention, the probe set can be immobilized on a substrate of a microarray. The oligonucleotide probe set immobilized on the microarray substrate is as described above.

본 발명의 방법에 있어서, 상기 식중독균의 검출은 검출 가능한 신호를 발생시키는 물질로 상기 시료의 게놈 DNA를 표지하고, 이를 상기 올리고뉴클레오티드 프로브와 혼성화시키고, 그로부터 발생하는 신호를 검출하여 이루어질 수 있다. 상기 검출 가능한 신호는 예를 들면, 광학적 신호 또는 전기적 신호가 될 수 있으나, 이들 예에 한정되는 것은 아니다. 상기 광학적 활성 물질은 형광 또는 인광을 발생시키는 물질일 수 있다. 상기 형광 물질은 예를 들면, 플루오로레신(fluorescein), Cy-5 및 Cy-3 일 수 있다. 또한, 상기 게놈 DNA는 혼성화 전 또는 후에 검출 가능한 신호를 발생시키는 물질로 표지되거나, 표지되어 있지 않을 수 있다. 표지되어 있지 않은 경우의 상기 게놈 DNA와 올리고뉴클레오티드 프로브의 혼성화 결과는 예를 들면, 전기적 신호를 통하여 검출할 수 있으나, 이들 예에 한정되는 것은 아니다.In the method of the present invention, the detection of food poisoning bacteria may be performed by labeling genomic DNA of the sample with a substance generating a detectable signal, hybridizing it with the oligonucleotide probe, and detecting a signal generated therefrom. The detectable signal may be, for example, an optical signal or an electrical signal, but is not limited to these examples. The optically active material may be a material that generates fluorescence or phosphorescence. The fluorescent material may be, for example, fluorescein, Cy-5, and Cy-3. In addition, the genomic DNA may or may not be labeled with a substance that generates a detectable signal before or after hybridization. The hybridization result of the genomic DNA and the oligonucleotide probe when not labeled can be detected by, for example, an electrical signal, but is not limited to these examples.

본 발명은 또한,The present invention also provides

본 발명의 일 실시예에 따른 하나 이상의 올리고뉴클레오티드 프로브 세트를 포함하는 식중독균 검출 키트를 제공한다. 올리고뉴클레오티드 프로브 세트는 전술한 바와 같다. 상기 키트는 또한 혼성화 반응에 필요한 시약을 필요로 하는데, 이는 당업계에 통상적으로 이용되는 것을 이용할 수 있으며, 특별히 제한되는 것은 아니다. 또한, 본 발명의 키트는 최적의 반응 수행 조건을 기재한 사용자 설명서를 추가로 포함할 수 있다.It provides a food poisoning bacteria detection kit comprising at least one oligonucleotide probe set according to an embodiment of the present invention. The oligonucleotide probe set is as described above. The kit also requires reagents for the hybridization reaction, which may be used conventionally in the art, and are not particularly limited. In addition, the kit of the present invention may further include a user manual describing the conditions for performing the optimal reaction.

이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

실시예Example

실험 방법Experiment method

사용한 미생물 균주Microbial Strains Used

본 발명에서 사용한 미생물은 표 1 과 같이 ATCC (the American Type Culture Collection) 균주들을 수집하여 사용하였다.The microorganism used in the present invention was used to collect the strains of the American Type Culture Collection (ATCC) as shown in Table 1.

표 1. 본 발명에 이용된 균주.Table 1. Strains used in the present invention.

Bell 균주Strain Bell 균주Strain Salmonella serovar Typhimurium Salmonella serovar Typhimurium ATCC 19585ATCC 19585 ListeriaListeria monocytogenesmonocytogenes ATCC 19118ATCC 19118 Salmonella serovar Typhimurium Salmonella serovar Typhimurium ATCC 14028ATCC 14028 ListeriaListeria monocytogenesmonocytogenes ATCC 7644ATCC 7644 Salmonella serovar Typhimurium Salmonella serovar Typhimurium ATCC 13311ATCC 13311 ListeriaListeria monocytogenesmonocytogenes ATCC 15313ATCC 15313 Salmonella serovar Typhi Salmonella serovar Typhi ATCC 6539ATCC 6539 ListeriaListeria ivanoviiivanovii ATCC 19119ATCC 19119 Salmonella serovar Typhi Salmonella serovar Typhi ATCC 33459ATCC 33459 ListeriaListeria welshimeriwelshimeri ATCC 35897ATCC 35897 Salmonella serovar Paratyphi B Salmonella serovar Paratyphi B ATCC 13428ATCC 13428 ListeriaListeria inocuainocua ATCC 33090ATCC 33090 Salmonella serovar Paratyphi C Salmonella serovar Paratyphi C ATCC 10719ATCC 10719 ListeriaListeria grayigrayi ATCC 25401ATCC 25401 Salmonella serovar Enteritidis Salmonella serovar Enteritidis ATCC 4931ATCC 4931 ListeriaListeria seeligeriseeligeri ATCC 35967ATCC 35967 SalmonellaSalmonella bongoribongori ATCC 43975ATCC 43975 StaphylococcusStaphylococcus aureusaureus ATCC 25923ATCC 25923 SalmonellaSalmonella entericaenterica salamaesalamae ATCC 15793ATCC 15793 StaphylococcusStaphylococcus aureusaureus ATCC 6538PATCC 6538P SalmonellaSalmonella entericaenterica arizonaearizonae ATCC 13314ATCC 13314 StaphylococcusStaphylococcus aureusaureus ATCC 6538ATCC 6538 SalmonellaSalmonella entericaenterica diarizonaediarizonae ATCC 43973ATCC 43973 StaphylococcusStaphylococcus aureusaureus ATCC 29737ATCC 29737 SalmonellaSalmonella entericaenterica houtenaehoutenae ATCC 43974ATCC 43974 StapylococcusStapylococcus haemolyticushaemolyticus ATCC 29970ATCC 29970 SalmonellaSalmonella entericaenterica indicaindica ATCC 43976ATCC 43976 StapylococcusStapylococcus epidermidisepidermidis ATCC 14990ATCC 14990 Salmonella serovar Choleraesuis Salmonella serovar Choleraesuis ATCC 13312ATCC 13312 VibrioVibrio parahaemolyticusparahaemolyticus ATCC 27969ATCC 27969 Salmonella serovar Gallinarum Salmonella serovar Gallinarum ATCC 9184ATCC 9184 VibrioVibrio parahaemolyticusparahaemolyticus ATCC 33844ATCC 33844 Salmonella serovar Pullorum Salmonella serovar pullorum ATCC 9120ATCC 9120 VibrioVibrio parahaemolyticusparahaemolyticus ATCC 17802ATCC 17802 Escherichia coli O157:H7 Esherichia coli O157: H7 ATCC 43894ATCC 43894 VibrioVibrio choleraecholerae ATCC 14547ATCC 14547 EscherichiaEsherichia colicoli ATCC 27325ATCC 27325 VibrioVibrio vulnificusvulnificus ATCC 33815ATCC 33815 EscherichiaEsherichia colicoli ATCC 23736ATCC 23736 CampylobacterCampylobacter jejunijejuni ATCC 43429ATCC 43429 EscherichiaEsherichia colicoli ATCC 25922ATCC 25922 CampylobacterCampylobacter jejunijejuni ATCC 33291ATCC 33291 EscherichiaEsherichia colicoli ATCC 11775ATCC 11775 ClostridiumClostridium botulinumbotulinum ATCC 3502ATCC 3502 YersiniaYersinia enterocoliticaenterocolitica ATCC 23715ATCC 23715 ClostridiumClostridium perfringensperfringens ATCC 3624ATCC 3624 BacillusBacillus cereuscereus ATCC11778ATCC11778 CitrobacterCitrobacter frenudiifrenudii ATCC 8090ATCC 8090 BacillusBacillus cereuscereus ATCC14579ATCC14579 EnterobacterEnterobacter aerogenesaerogenes ATCC 13048ATCC 13048 BacillusBacillus cereuscereus ATCC10876ATCC10876 EnterobacterEnterobacter cloacaecloacae ATCC 13047ATCC 13047 BacillusBacillus subtilissubtilis ATCC 6633ATCC 6633 EnterobacterEnterobacter sakazakiisakazakii ATCC 29544ATCC 29544 BacillusBacillus subtilissubtilis ATCC 6051ATCC 6051 EnterococcusEnterococcus faecalisfaecalis ATCC 19433ATCC 19433 BacillusBacillus thuringiensisthuringiensis ATCC 35646ATCC 35646 Kelbsiella pneumoniea subsp. pneumoniae Kelbsiella pneumoniea subsp. pneumoniae ATCC 8724ATCC 8724 BacillusBacillus thuringiensisthuringiensis ATCC 10792ATCC 10792 ProteusProteus vulgarisvulgaris ATCC 29905ATCC 29905 BacillusBacillus anthracisanthracis ATCC 14578ATCC 14578 RahnellaRahnella aquatilisaquatilis ATCC 15552ATCC 15552 ListeriaListeria monocytogenesmonocytogenes ATCC 19111ATCC 19111 ShigellaShigella boydiiboydii ATCC 8700ATCC 8700 ListeriaListeria monocytogenesmonocytogenes ATCC 19113ATCC 19113 ShigellaShigella flexneriflexneri ATCC 12022ATCC 12022 ListeriaListeria monocytogenesmonocytogenes ATCC 19114ATCC 19114 ShigellaShigella sonneisonnei ATCC 25931ATCC 25931 ListeriaListeria monocytogenesmonocytogenes ATCC 19115ATCC 19115

총 69개의 균주들이 사용되었으며 식중독균들 및 비 식중독균들로 이루어져 있다. 사용되어진 식중독균들은 다음과 같다: 바실러스 세레우스(Bacillus cereus), 리스테리아 모노시토게네스(Listeria monocytogenes), 스태필로코커스 오레우스(Staphylococcus aureus), 클로스트리디움 보툴리눔(Clostridium botulinum), 클로스트리디움 퍼프린젠스(Clostridium perfringens), 캄필로박터 제주니(Campylobacter jejuni), 비브리오 파라해모리티쿠스(Vibrio parahaemolyticus), 여시니아 엔테로콜리티카(Yersinia enterocolitica), 에세리키아 콜라이(Escherichia coli) O157:H7, 살모넬라 엔테리카 세로바 티피(Salmonella enterica serovar Typhi) 및 살모넬라 엔테리카 세로바 티피무리움(Salmonella enterica serovar Typhimurium). 각각의 미생물들은 각각의 배지와 조건에서 배양하였으며, Bacillus anthracis ATCC 14578와 Clostridium botulinum ATCC 3502 의 경우는 한국국립보건원 (the Korea Center for Disease Control and Prevention, KCDC)에서 게놈 DNA를 얻었다.A total of 69 strains were used and consisted of food poisoning bacteria and non-food poisoning bacteria. Food poisoning bacteria used were as follows: Bacillus cereus , Listeria monocytogenes ( Listeria) monocytogenes ), Staphylococcus aureus), Clostridium botulinum (Clostridium botulinum ), Clostridium perfringens ), Campylobacter jejuni ), Vibrio parahaemoritis ( Vibrio) parahaemolyticus ), Yersinia enterocolitica ), Escherichia coli ) O157: H7, Salmonella enterica serovatipi ( Salmonella) enterica serovar Typhi) and Salmonella typhimurium Entebbe Rica vertical bar (Salmonella enterica serovar Typhimurium). Each microorganism was incubated in each medium and condition, Bacillus anthracis ATCC 14578 and Clostridium In the case of botulinum ATCC 3502, genomic DNA was obtained from the Korea Center for Disease Control and Prevention (KCDC).

게놈 Genome DNADNA 추출 extraction

각각의 키운 미생물들을 원심 분리하여 균체를 모은 뒤 DNeasy Tissue Kit (QIAGEN, Hilden, Germany)을 이용하여 게놈 DNA를 추출하였다. 추출한 게놈 DNA의 농도는 UV-spectrophotometer (Model UV-1700, Shimadzu, Tokyo, Japan)를 이용하여 측정하였으며 흡광도의 비 (A260/A280)가 1.8에서 2 사이의 DNA 들만을 본 발명에 사용하였다.Each grown microorganism was centrifuged to collect the cells and genomic DNA was extracted using DNeasy Tissue Kit (QIAGEN, Hilden, Germany). The concentration of extracted genomic DNA was measured using a UV-spectrophotometer (Model UV-1700, Shimadzu, Tokyo, Japan), and only the DNAs having an absorbance ratio (A 260 / A 280 ) of 1.8 to 2 were used in the present invention. .

식중독균들의 게놈 Genome of food poisoning bacteria DNADNA 서열 order

본 발명에서 사용된 게놈 DNA 서열들은 표 2와 같다.Genomic DNA sequences used in the present invention are shown in Table 2.

표 2. 본 발명에 이용된 게놈 서열.TABLE 2 Genome sequences used in the present invention.

박테리아 균주Bacterial strains Ref. seqRef. seq GenbankGenbank Total base(bp)Total base (bp) 단백질 코딩 유전자Protein coding genes 병원성Pathogenic Bacillus cereus ATCC 14579 Bacillus cereus ATCC 14579 NC_004722 NC_004722 AE016877 AE016877 5,411,8095,411,809 52345234 병원균Pathogens Listeria monocytogenes strain EGD Listeria monocytogenes strain EGD NC_003210 NC_003210 AL591824 AL591824 2,944,5282,944,528 28462846 병원균Pathogens ListeriaListeria inocuainocua NC_003212NC_003212 AL592022 AL592022 3,011,2083,011,208 29892989 비병원균Non-pathogenic bacteria Stapylococcus aureus N315 Stapylococcus aureus N315 NC_002745NC_002745 BA000018BA000018 2,814,8162,814,816 25942594 병원균Pathogens Stapylococcus aureus Mu50 Stapylococcus aureus Mu50 NC_002758 NC_002758 BA000017 BA000017 2,878,5292,878,529 27142714 병원균Pathogens Stapylococcus aureus MW2 Stapylococcus aureus MW2 NC_003923 NC_003923 BA000033BA000033 2,820,4622,820,462 26322632 병원균Pathogens Clostridium botulinum Hall strain A (ATCC 3502) Clostridium botulinum Hall strain A (ATCC 3502) CB.dbsCB.dbs 3,886,9163,886,916 병원균Pathogens Clostridium perfringens str. 13 Clostridium perfringens str. 13 NC_003366 NC_003366 BA000016 BA000016 3,031,4303,031,430 26602660 병원균Pathogens Campylobacter jejuni subsp. jejuni NCTC 11168 Campylobacter jejuni subsp. jejuni NCTC 11168 NC_002163NC_002163 AL111168 AL111168 1,641,4811,641,481 16341634 병원균Pathogens Vibrio parahaemolyticcus RIMD 2210633 Vibrio parahaemolyticcus RIMD 2210633 NC_004603 NC_004603 BA000031 BA000031 3,288,5583,288,558 30803080 병원균Pathogens NC_004605 NC_004605 BA000032 BA000032 1,877,2121,877,212 17521752 YersiniaYersinia enterocoliticaenterocolitica YE.dbsYE.dbs 4,615,8994,615,899 병원균Pathogens Escherichia coli O157:H7 EDL933 Esherichia coli O157: H7 EDL933 NC_002655 NC_002655 AE005174 AE005174 5,528,4455,528,445 53245324 병원균Pathogens Escherichia coli O157:H7 Esherichia coli O157: H7 NC_002695 NC_002695 BA000007 BA000007 5,498,4505,498,450 53615361 병원균Pathogens Escherichia coli K12 Esherichia coli K12 NC_000913NC_000913 U00096U00096 4,639,2214,639,221 42794279 비병원균Non-pathogenic bacteria Salmonella enterica serovar Typhimurium LT2 Salmonella enterica serovar Typhimurium LT2 NC_003197 NC_003197 AE006468 AE006468 4,857,4324,857,432 44514451 병원균Pathogens Salmonella enterica serovar Typhi CT18 Salmonella enterica serovar Typhi CT18 NC_003198NC_003198 AL513382 AL513382 4,809,0374,809,037 43954395 병원균Pathogens

본 발명에 사용된 대부분의 식중독균 서열 파일 (ffn file (코딩 영역 서열 ), fna file (전체 게놈 서열))들은 NCBI에서 얻었으며, Yersinia enterocoliticaClostridium botulinum Hall strain A (ATCC 3502)의 게놈 DNA 서열들은 Sanger Institute (http://www.sanger.ac.uk/Projects/Y_enterocolitica/, http://www.sanger.ac.uk/Projects/C_botulinum/) 에서 얻었다. 식중독균들과 관련이 있는 비병원성의 균들의 게놈 DNA 서열들도 사용되었으며, 총 16개의 게놈 DNA 서열들이 본 발명에서 사용되었다.Most of the food poisoning sequence files used in the present invention (ffn file (coding region sequence), fna file (whole genome sequence)) were obtained from NCBI, Yersinia enterocolitica and Clostridium Genomic DNA sequences of botulinum Hall strain A (ATCC 3502) are described in Sanger Institute ( http://www.sanger.ac.uk/Projects/Y_enterocolitica/ , http://www.sanger.ac.uk/Projects/C_botulinum/ ). Obtained from Genomic DNA sequences of non-pathogenic bacteria associated with food poisoning bacteria were also used, and a total of 16 genomic DNA sequences were used in the present invention.

비교 유전체학을 이용한 식중독균 특이적인 Food Poisoning Bacteria Specific Using Comparative Genomics 프로브Probe 선별 작업 Screening

각 식중독균에 특이적인 70-mer 올리고뉴클레오티드들의 선별 작업에 대한 모식도는 도 1과 같다. 16개의 박테리아의 게놈 DNA 서열들의 데이터베이스를 만들었으며, 각각의 11개의 식중독균들의 코딩 영역 서열들은 BLAST (Basic Local Alignment Search Tool) 프로그램을 이용하여 게놈 DNA 서열들의 데이터베이스에 비교하였다. Clostridium botulinumYersinia enterocolitica 의 게놈 DNA의 경우에는 유전자 서열 부분이 아직까지 분석이 되어있지 않기 때문에 전체 게놈 서열들을 800bp 크기로 400bp씩 중복되도록 자른 뒤 800bp 크기의 DNA 조각들을 게놈 DNA 서열들의 데이터베이스와 비교하였다.Schematic diagram of the screening of the 70-mer oligonucleotides specific to each food poisoning bacteria is shown in FIG. A database of 16 bacterial genomic DNA sequences was made, and the coding region sequences of each of the 11 food poisoning bacteria were compared to a database of genomic DNA sequences using the BLAST (Basic Local Alignment Search Tool) program. Clostridium In the genomic DNA of botulinum and Yersinia enterocolitica , since the gene sequence portion has not been analyzed yet, the entire genome sequences were cut into 400 bp by 800 bp, and then 800 bp DNA fragments were compared with a database of genomic DNA sequences.

BLAST 결과물 중, 각각의 유전자 (혹은 각각의 DNA 조각들)에 대하여 유사도가 가장 높은 결과물들을 선택하였으며, 이 결과들에 기초하여 각각의 식중독균에 특이적일 것으로 생각되는 유전자 (혹은 DNA 조각)들을 선별하였으며, 다시 BLAST 프로그램을 이용하여 NCBI의 nr 데이타베이스와 비교하였다. 이 방법을 통하여 각 각의 식중독균에 특이적일 것으로 생각되는 유전자들을 선별하였으며 (표 3), 선별된 유전자들은 시작 서열부터 1 base pair의 간격으로 이동하며 70-mer 크기의 DNA 조각들로 나누었다. 이들 70-mer DNA 조각들 중 Tm (melting temperature) 값이 78±0.5℃인 서열들을 다시 선별하였으며 데이타베이스 들과 다시 비교하여 각각의 식중독균에 특이적인 70-mer DNA 서열들을 최종적으로 선별하였다.Of the BLAST results, the products with the highest similarity were selected for each gene (or individual DNA fragments), and based on these results, genes (or DNA fragments) that were thought to be specific for each food poisoning bacterium were selected. Again, it was compared with NCBI's nr database using the BLAST program. Through this method, genes thought to be specific for each food poisoning bacterium were selected (Table 3), and the selected genes were divided into 70-mer size DNA fragments shifted by 1 base pair from the starting sequence. Of these 70-mer DNA fragments, the sequences with Tm (melting temperature) value of 78 ± 0.5 ° C were reselected and 70-mer DNA sequences specific for each food poisoning bacterium were finally selected by comparison with the databases.

표 3. 11가지 식중독균의 게놈 DNA 서열로부터 선발된 70-mer 프로브 및 구축된 70-mer 올리고뉴클레오티드의 수.Table 3. Number of 70-mer probes and constructed 70-mer oligonucleotides selected from genomic DNA sequences of 11 food poisoning bacteria.

Bacillus cereus ATCC 14579 Bacillus cereus ATCC 14579 Listeria monocytogenes EGD Listeria monocytogenes EGD Stapylococcus aureus Mu50 Stapylococcus aureus Mu50 Clostridium botulinum Hall strain A Clostridium botulinum Hall strain A Clostridium perfringens str. 13 Clostridium perfringens str. 13 CampylobacterCampylobacter jejuni NCTC 11168 jejuni NCTC 11168 유전자 수 (단편)Gene count (fragment) 5,2345,234 2,8462,846 2,7142,714 9758a 9758 a 2,6602,660 1,6341,634 1차 선발된 유전자 수Number of genes selected first 2,5082,508 136136 1,4221,422 3402a 3402 a 1,4141,414 935935 2차 선발된 유전자 수Secondary Selected Gene Count 2,0042,004 113113 333333 38a 38 a 916916 722722 2차 선발된 유전자에서 70mer DNA 단편 수Number of 70mer DNA Fragments in Secondary Selected Genes 1,110,0151,110,015 81,91981,919 252,300252,300 27,77827,778 637,562637,562 524,884524,884 70mer DNA 단편 수 Tm.=78℃70mer DNA Fragment Number Tm. = 78 ° C 10,87310,873 805805 1,9881,988 251251 1,2011,201 2,4102,410 BLAST 결과를 이용하여 선발된 단편 수Number of fragments selected using BLAST results 4949 152152 364364 4444 228228 238238 최종 선발된 단편 수Number of pieces selected 3636 106106 126126 1717 103103 122122 구축된 70-mer 올리고뉴클레 오티드 수Constructed 70-mer oligonucleotides 1919 3030 2020 1515 3030 3030 FBP-1 구축에 이용된 70mer 프로브 수Number of 70mer Probes Used to Build FBP-1 1010 1010 1010 1010 1010 1010

(계속)(continue)

Vibrio parahaemolyt icus RIMD Vibrio parahaemolyt icus RIMD YersiniaYersinia enterocoliticaenterocolitica Escherichia coli O157:H7 EDL933 Escherichia coli O157: H7 EDL933 Salmonella enterica serovar Typhimurium LT2 Salmonella enterica serovar Typhimurium LT2 Salmonella enterica serovar Typhi CT18 Salmonella enterica serovar Typhi CT18 유전자 수 (단편)Gene count (fragment) 4,8314,831 11539a 11539 a 5,3235,323 4,4514,451 4,3954,395 1차 선발된 유 전자 수Primary Selected Gene Count 2,6532,653 2948a 2948 a 848848 196196 290290 2차 선발된 유 전자 수Secondary Selected Gene Count 1,6691,669 87a 87 a 336336 138138 210210 2차 선발된 유 전자에서 70mer DNA 단편 수Number of 70mer DNA Fragments in Secondary Selected Genes 1,069,4841,069,484 63,77163,771 166,599166,599 97,85097,850 97,87597,875 70mer DNA 단편 수 Tm.=78℃70mer DNA Fragment Number Tm. = 78 ° C 8,8648,864 332332 1,1141,114 518518 598598 BLAST 결과를 이용하여 선발 된 단편 수Number of fragments selected using BLAST results 1,5711,571 6767 141141 124124 166166 최종 선발된 단 편 수Final Selected Shorts 2626 2424 6262 9191 102102 구축된 70-mer 올리고뉴클레 오티드 수Constructed 70-mer oligonucleotides 2020 2020 1515 4141 3535 FBP-1 구축에 이용된 70mer 프로브 수Number of 70mer Probes Used to Build FBP-1 1010 1010 1010 1010 1010

양성 대조군 및 음성 대조군의 제작Construction of Positive Control and Negative Control

애기장대(Arabidopsis thaliana)의 putative tubulin beta-4 chain (accession number AY059075)과 actin 3 (accession number AY093784) 유전자의 일부분을 PCR을 이용하여 하기 프라이머로 증폭하였다: Actin3-f 5'-CTCTTGACTACGAGCAGGAA-3'(서열번호 113) 및 Actin3-r 5'-AGTCCTTCCTGATGTCGACA-3'(서열번호 114); Tb4-f 5'-AAGAGGTTGACGAGCAGATG-3'(서열번호 115) 및 Tb4-r 5'-GCCTTTCTCCTGAACATAGC-3'(서열번호 116). 증폭된 DNA 조각은 Perfect-T 클로닝 키트 (TaKaRa, Shiga, Japan)을 이용하여 pMD18-T 벡터에 삽입되었으며 제작된 플라 스미드 DNA는 -20℃에 저장하였다. Arabidopsis A portion of the putative tubulin beta-4 chain (accession number AY059075) and actin 3 (accession number AY093784) genes of thaliana were amplified by PCR with the following primers: Actin3-f 5'-CTCTTGACTACGAGCAGGAA-3 '(SEQ ID NO: 113) ) And Actin3-r 5'-AGTCCTTCCTGATGTCGACA-3 '(SEQ ID NO: 114); Tb4-f 5'-AAGAGGTTGACGAGCAGATG-3 '(SEQ ID NO: 115) and Tb4-r 5'-GCCTTTCTCCTGAACATAGC-3' (SEQ ID NO: 116). The amplified DNA fragment was inserted into pMD18-T vector using Perfect-T cloning kit (TaKaRa, Shiga, Japan), and the produced plasmid DNA was stored at -20 ° C.

양성 대조군 및 음성 대조군으로 사용하기 위한 프로브의 서열은 각각A rabido psis thaliana의 putative tubulin beta-4 chain (accession number AY059075)과 actin 3 gene (accession number AY093784)으로부터 특이적인 서열을 선별하였다: 5'-AAGTCCAGTGTCTGTGATATTGCACCAAAGGGTTTGAAAATGGCGTCTACTTTCATTGGTAACTCAACCT-3'(서열번호 111) 및 5'-AGGTTCTTTACCAGCCATCTATGATTGGTATGGAGAATGCTGGTATCCATGAAACCACCTATAACTCCAT-3'(서열번호 112).Sequences of the probes for use as positive and negative controls were selected from the putative tubulin beta-4 chain (accession number AY059075) and actin 3 gene (accession number AY093784) of A rabido psis thaliana , respectively: 5'- AAGTCCAGTGTCTGTGATATTGCACCAAAGGGTTTGAAAATGGCGTCTACTTTCATTGGTAACTCAACCT-3 '(SEQ ID NO: 111) and 5'-AGGTTCTTTACCAGCCATCTATGATTGGTATGGAGAATGCTGGTATCCATGAAACCACCTATAACTCCAT-3'.

올리고뉴클레오티드 Oligonucleotide 마이크로어레이Microarray 제작 making

112개의 프로브들(224 올리고뉴클레오티드 DNA 스팟)로 구성된 올리고뉴클레오티드 DNA 칩을 제작하였다. 5'말단을 아민으로 변환시킨 70-mer 올리고뉴클레오티드들은 Illumina 사(Illumina Inc., San Diego, CA)에서 제작하였으며, 50pmol의 농도로 혼성화 용액에 녹인 후, CMT-GAPS II 실란 슬라이드 글라스 위에 pixsys 5500 arrayer (Cartesian Technologies, USA)를 이용하여 DNA 칩을 제작하였다. 제작이 끝난 뒤에는 하나의 칩을 Syto61으로 염색한 후, GenePix 4100A scanner (Axon Instruments, Redwood City, CA)로 스캔하여 칩의 제작을 확인하였다. 칩 위의 각각의 블록은 혼성화 바로 전에 CoverWell perfusion chamber (PC8R-0.5, Grace-Bio Labs, Inc., Bend, OR)로 덮어서 구역을 구분하였다. An oligonucleotide DNA chip consisting of 112 probes (224 oligonucleotide DNA spots) was constructed. The 70-mer oligonucleotides converting the 5 'end to an amine were prepared by Illumina (Illumina Inc., San Diego, Calif.), Dissolved in a hybridization solution at a concentration of 50 pmol, and then pixsys 5500 on CMT-GAPS II silane slide glass. DNA chips were prepared using an arrayer (Cartesian Technologies, USA). After fabrication, one chip was dyed with Syto61 and scanned with a GenePix 4100A scanner (Axon Instruments, Redwood City, Calif.) To confirm the fabrication of the chip. Each block on the chip was zoned by covering with a CoverWell perfusion chamber (PC8R-0.5, Grace-Bio Labs, Inc., Bend, OR) just before hybridization.

시료 sample DNADNA of Cy3Cy3 형광물질 표지 및  Fluorescent material labeling and 혼성화Hybridization

미생물의 게놈 DNA들은 BioPrime DNA labeling System (Invitrogen, Carlsbad, CA)를 이용하여 Cy3 형광물질로 표지하였다. 500ng 의 게놈 DNA 와 1ng 의 양성 대조군을 eppendorf 튜브에 넣고 3차 증류수를 넣어 20 ㎕까지 채운 뒤 20 ㎕의 2.5X Random Primers Solution을 넣고 끓는 물에 5분 동안 넣은 뒤, 곧바로 얼음 속에 넣어 온도를 낮추었다. 6 ㎕의 3차 증류수, 1 ㎕의 dNTP 용액 (10 mM dATP, dGTP 및 dTTP; 3 mM dCTP), 2 ㎕의 Cy3-dCTP (1 mM, GeneChem Inc., Daejeon, Korea), 40 유닛의 Klenow fragment를 넣은 뒤, 37℃에서 2시간 동안 반응을 진행하였다. 반응이 끝난 뒤, Cy3 형광물질로 표지된 DNA는 소듐 아세테이트 (3M, pH 5.2)와 100% 에탄올로 침전시킨 뒤, 45 ㎕ 혼성화 용액으로 녹였다. 이 용액은 2분간 끓는 물에서 끓인 뒤, 상온에서 식히고 마이크로어레이 칩의 각각의 챔버에 넣고 60℃에서 8시간 동안 혼성화하였다. 혼성화가 끝난 뒤, DNA 칩을 1차 세정액 (2X SSC, 0.1% SDS)으로 60℃에서 5분간 세정하고, 2차 세정액 (1X SSC)으로 실온에서 3분간 세정한 후, 3차 세정액 (0.2X SSC)으로 실온에서 3분간 반응하고 원심 분리하여 건조시켰다.The microbial genomic DNAs were labeled with Cy3 fluorescent material using the BioPrime DNA labeling system (Invitrogen, Carlsbad, Calif.). 500 ng of genomic DNA and 1 ng of positive control were added to eppendorf tube and filled with 20 μl of tertiary distilled water. Then, 20 μl of 2.5X Random Primers Solution was added to boiling water for 5 minutes. It was. 6 μl of tertiary distilled water, 1 μl of dNTP solution (10 mM dATP, dGTP and dTTP; 3 mM dCTP), 2 μl of Cy3-dCTP (1 mM, GeneChem Inc., Daejeon, Korea), 40 units of Klenow fragment After the addition, the reaction was carried out for 2 hours at 37 ℃. After the reaction, DNA labeled with Cy3 fluorescent material was precipitated with sodium acetate (3M, pH 5.2) and 100% ethanol, and then dissolved in 45 μl hybridization solution. The solution was boiled in boiling water for 2 minutes, cooled at room temperature and placed in each chamber of the microarray chip and hybridized at 60 ° C. for 8 hours. After hybridization, the DNA chip was washed for 5 minutes at 60 ° C. with a primary cleaning solution (2X SSC, 0.1% SDS), and for 3 minutes at room temperature with a secondary cleaning solution (1X SSC), followed by a tertiary cleaning solution (0.2X SSC) at room temperature for 3 minutes and centrifuged to dryness.

스캔, 이미지 분석 및 분류Scan, image analysis and classification

혼성화가 끝난 칩은 Axon Instruments GenePix 4100A scanner (Axon Instruments, Redwood City, CA)를 이용하여 5 ㎛의 해상도로 스캔을 하였으며, 스 캔한 이미지는 GenePix Pro 6.0 (Axon Instruments, Redwood City, CA) 프로그램을 이용하여 분석하였다. 이미지 분석된 파일들은 MS excel 파일로 저장하였다. 각각의 미생물에 대한 분석 MS excel 파일들은 GeneSpring 7.2 (Silicon Genetics, Palo Alto, CA) 프로그램을 이용하여 분석하였다. 사용된 알고리즘은 Pearson correlation에 기본을 두어 분석하였다.The hybridized chip was scanned at a resolution of 5 μm using an Axon Instruments GenePix 4100A scanner (Axon Instruments, Redwood City, CA), and the scanned image was scanned using GenePix Pro 6.0 (Axon Instruments, Redwood City, CA) program. And analyzed. Image analyzed files were saved as MS Excel file. Analysis of each microorganism MS excel files were analyzed using GeneSpring 7.2 (Silicon Genetics, Palo Alto, CA) program. The algorithm used was analyzed based on Pearson correlation.

실시예Example 1: 비교 유전체학을 통한 11개 식중독균에 대한 특이적인  1: Specific for 11 food poisoning bacteria through comparative genomics 프로브Probe 선별 및  Screening and FBPFBP -1의 제작-1 made

비교유전체학을 통하여 11개 식중독균에 특이적인 70-mer 올리고뉴클레오티드들을 선별하였으며, 그 과정이 표 3에 나타나 있다. 11개의 식중독균에서 총 275개의 70mer의 프로브들이 제작되었다. 이 프로브들은 슬라이드 글라스에 고정되었으며, 다양한 미생물의 게놈 DNA로부터 준비된 시료들과 반응하였다. 275개의 프로브들 중 최종적으로 110개의 프로브들(각각의 식중독균에 대하여 10개의 프로브)이 선발되었으며, 도 2와 같이 FBP-1이라고 명명한 마이크로어레이 칩을 제작하였다. 제작된 FBP-1은 총 8개의 블록으로 나누어져 있으며, 각각의 블록은 110개의 식중독균 특이적 프로브들이 2번씩 반복으로 찍혀있으며, 양성 대조군 및 음성 대조군이 찍혀 있다. 그러므로 제작된 FBP-1은 한 번의 실험으로 총 8개의 시료를 반응할 수 있으며, 각각의 블록은 12줄로서 각 줄은 20개의 스팟(10 개의 프로브)으로 이루어져 있다.Comparative genetics was used to select 70-mer oligonucleotides specific for 11 food poisoning bacteria, and the process is shown in Table 3. A total of 275 70mer probes were prepared from 11 food poisoning bacteria. These probes were fixed on slide glass and reacted with samples prepared from genomic DNA of various microorganisms. Finally, 110 probes (10 probes for each food poisoning bacterium) of 275 probes were selected, and a microarray chip named FBP-1 was manufactured as shown in FIG. 2. The produced FBP-1 is divided into a total of eight blocks, each block is repeated twice with 110 phagocytic bacteria-specific probes, a positive control and a negative control. Therefore, the manufactured FBP-1 can react a total of eight samples in one experiment. Each block is composed of 12 rows and each row consists of 20 spots (10 probes).

표 4. FBP-1의 구축을 위한 70-mer 올리고뉴클레오티드 프로브 서열.Table 4. 70-mer oligonucleotide probe sequences for construction of FBP-1.

Figure 112008038435230-PAT00001
Figure 112008038435230-PAT00001

Figure 112008038435230-PAT00002
Figure 112008038435230-PAT00002

Figure 112008038435230-PAT00003
Figure 112008038435230-PAT00003

Figure 112008038435230-PAT00004
Figure 112008038435230-PAT00004

Figure 112008038435230-PAT00005
Figure 112008038435230-PAT00005

식중독균들의 확보된 프로브 (70mer)를 이용하여 DNA 칩을 도 2와 같이 제작하였다. 각각의 식중독균에 대하여 10개씩의 70mer 프로브 (5'-아민 변형)를 제작하였으며, 제작된 프로브의 농도를 50uM로 맞추어서 아민-실란 코팅된 슬라이드 (corining) 위에 프린트하여 일차적으로 DNA 칩을 제작하였다.DNA chips were prepared as shown in FIG. 2 using the probes (70mer) obtained from food poisoning bacteria. Ten 70mer probes (5'-amine modifications) were prepared for each food poisoning bacterium, and DNA chips were prepared primarily by printing onto amine-silane coated slides at a concentration of 50 uM.

제작된 DNA 칩은 식중독균 10 종류에 대한 특이적인 프로브들이 프린트되어 있으며, 인간에게 특이적으로 질병을 일으키는 S. typhi에 특이적인 프로브 10개를 프린트하여 총 110개의 스팟과 2개의 양성 대조군 스팟 및 2개의 음성 대조군 스팟을 포함하고 있다.The fabricated DNA chip was printed with 10 specific probes for 10 kinds of food poisoning bacteria, and a total of 110 spots, 2 positive control spots, and 2 prints of 10 probes specific for S. typhi that specifically caused diseases in humans. Negative control spots.

선별한 각 식중독균에 특이적인 프로브들과 실험결과를 토대로 하여 최종적으로 110개의 스팟 (각 식중독균에 대하여 10개씩의 특이적인 프로브를 선별하였음)이 찍혀있는 도 2와 같은 마이크로어레이 칩을 제작하였으며, FBP-1 (Food Borne Pathogen)이라고 명명하였다.Based on the probes specific to the selected food poisoning bacteria and the experimental results, a microarray chip as shown in FIG. 2 having 110 spots (selected 10 specific probes for each food poisoning bacteria) was finally manufactured. -1 (Food Borne Pathogen).

최종적으로 제작된 DNA 칩은 총 8개의 블록으로 이루어져 있으며, 각 블록은 110개의 프로브들이 두 번 반복으로 찍혀 있으며, 양성 대조군 및 음성 대조군 용으로 각각 식물에 존재하는 Tubulin beta-4 chain과 Actin 3 유전자의 서열을 사용하였다.The final DNA chip consists of a total of eight blocks, each of which has 110 probes repeated twice, and the tubulin beta-4 chain and Actin 3 genes present in the plants for the positive control and the negative control, respectively. The sequence of was used.

게놈 DNA는 Bioprime DNA labeling kit을 사용하였으며 Cy3로 표지하였다. 양성 대조군 DNA로는 식물체의 Tubulin beta-4 chain 유전자를 플라스미드 DNA에 삽입하여 사용하였다.Genomic DNA was used for Bioprime DNA labeling kit and labeled with Cy3. As a positive control DNA, the tubulin beta-4 chain gene of the plant was inserted into the plasmid DNA.

실시예Example 2:  2: 엔테로박테리아(Enterobacteriae)에Enterobacteriae 속하는 식중독균들의  Of food poisoning bacteria 마이크로어레이Microarray 결과 result

Salmonella, Escherichia coli O157:H7 및 Yersinia enterocolitica 는 Enterobacteriae에 속해 있는 식중독균으로 알려져 있다. 이러한 식중독균들은 Enterobacteriae에 속하는 다른 미생물들과의 게놈 DNA 서열의 유사도가 높기 때문에 특이적인 검출이 힘든 것으로 알려져 있다. 최근에 Escherichia coli, ShigellaSalmonella 를 동정하기 위하여 gyrB 유전자로부터 제작된 10개의 프로브들이 평가되었다 (Kakinuma et al. 2003. Biotechnology and Bioengineering. 83: 721-728). 그러나 몇몇의 프로브들은 다른 종류의 미생물들과 반응하는 결과를 나타내었으며, 그러므로 Enterobacteriae 내에 속하는 미생물들과 구분할 수 있는 충분한 결과를 보여주지 못하였다. 본 발명에서는 Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Typhi, Escherichia coli O157:H7 와 Yersinia enterocolitica에 특이적인 프로브들을 제시하였으며, 각각의 10개의 프로브들은 도 3과 같이 각각의 식중독균에 특이적인 것을 확인하였다. Salmonella enterica serovar Typhimurium은 도 3 (A, B, C)과 같이 10 개의 선별된 프로브에 특이적으로 반응하는 것을 확인하였다.Salmonella, Escherichia coli O157: H7 and Yersinia enterocolitica is known as a food poisoning bacterium belonging to Enterobacteria. These food poisoning bacteria are known to be difficult to detect because they have high similarity in genomic DNA sequence with other microorganisms of Enterobacteria. Recently 10 probes constructed from the gyrB gene were evaluated to identify Escherichia coli , Shigella and Salmonella (Kakinuma et al. 2003. Biotechnology and Bioengineering. 83: 721-728). However, some probes showed reaction with other microorganisms, and thus did not show enough results to distinguish them from microorganisms within Enterobacteriae. Salmonella in the present invention enterica serovar Typhimurium, Salmonella enterica serovar Typhi, Escherichia coli O157: H7 with Yersinia Probes specific to enterocolitica were presented, and each of the ten probes was confirmed to be specific to each food poisoning bacterium as shown in FIG. 3. Salmonella enterica serovar Typhimurium was confirmed to specifically react to 10 selected probes as shown in Figure 3 (A, B, C).

10개의 Salmonella enterica serovar Typhimurium 특이적인 프로브들 중 4개의 프로브는 포유동물이나 조류에 특이적으로 질병을 일으키는 것으로 알려진 Salmonella subspecies I 에 속하는 serovar들에도 특이적으로 반응하는 것을 확인할 수 있었다 (도 7, Salmonella serovar Choleraesuis, Enteritidis, Pullorum, Gallinarum, Paratyphi C and Paratyphi B). Salmonella subspecies I 에 속하는 않는 Salmonella의 경우에는 어떠한 프로브와도 반응하지 않는 것을 확인할 수 있었다. Salmonella serovar Typhi에 특이적인 프로브의 경우, Salmonella serovar Typhi ATCC 33459와 ATCC 6539가 도 3 과 도 7과 같이 특이적으로 반응하는 것을 확인할 수 있었다. Salmonella serovar Paratyphi A 는 인간에 특이적으로 질병을 일으키는 것으로 알려져 있는 균주로서 역시 serovar Typhi에 특이적인 프로브들 가운데 3개의 프로브와 반응하는 것을 확인할 수 있었으며 이는 기존에 보고되어진 것과 같이 serovar Typhi 와 Paratyphi A의 유전적인 형질이 가까운 것을 확인할 수 있는 결과였다(데이타 미제시). Yersinia enterocolitica ATCC 23715와 Escherichia coli O157:H7 ATCC 43894의 경우에도 도 3 (G 및 E)과 같이 각각의 특이적인 프로브들과 반응하는 것을 확인할 수 있었다.Of the 10 Salmonella enterica serovar Typhimurium-specific probes, four probes were found to react specifically to serovars belonging to Salmonella subspecies I, which are known to cause disease in mammals or birds (Fig. 7, Salmonella). serovar Choleraesuis, Enteritidis, Pullorum, Gallinarum, Paratyphi C and Paratyphi B). If Salmonella in that belonging to Salmonella subspecies I has been confirmed that it does not react with any probe. In the case of Salmonella serovar Typhi specific probes, Salmonella serovar Typhi ATCC 33459 and ATCC 6539 reacted specifically as shown in FIGS. 3 and 7. Salmonella serovar Paratyphi A is a strain that is known to cause disease in humans, and it was confirmed that it reacts with three of serovar Typhi-specific probes, which has been reported as serovar Typhi and Paratyphi A. Genetic traits were confirmed to be close (data not shown). Yersinia enterocolitica ATCC 23715 and Escherichia In the case of coli O157: H7 ATCC 43894, as shown in FIG. 3 (G and E), it was confirmed that the reaction with each specific probe.

특히, 도 3 (F) 및 도 7과 같이 비병원성 균인 Escherichia coli 들이 반응하지 않는 것을 확인함으로써, 본 발명에서 선별한 프로브들은 병원성의 Escherichia coli O157:H7 균주와 비병원성 Escherichia coli를 선별할 수 있는 능력을 갖고 있는 것을 확인할 수 있었다. Enterobacteriae에 속하는 다수의 비병원성 균들과 반응한 결과, 도 7과 같이 식중독균에 특이적인 것을 확인할 수 있었다.In particular, Escherichia, a non-pathogenic bacterium as shown in FIGS. 3F and 7 coli By confirming that they do not react, the probes selected in the present invention are pathogenic Escherichia coli O157: H7 strain and non-pathogenic Escherichia. Coli was selected to have the ability to screen. As a result of reacting with a number of non-pathogenic bacteria belonging to Enterobacteriae, it was confirmed that the specific to the food poisoning bacteria as shown in FIG.

E. coli O157 : H7: E. coli O157:H7 ATCC 43894의 대하여 특이적으로 반응하는 것으로 확인하였으며, 병원성을 갖지 않는 E. coli  균주들에 대하여는 반응하지 않는 것을 확인하였다. E. coli O157: H7: E. coli O157: were found to specifically react against the H7 ATCC 43894, was found to having no pathogenicity which do not react with respect to the E. coli strains.

Salmonella typhimurium , Salmonella typhi : Salmonella typhimurium , Salmonella typhi에 대하여 특이적으로 반응하였으나, 다양한 Salmonella 균주들과 반응한 결과 Salmonella serovar에 따라 다양한 패턴의 마이크로어레이 실험결과가 나왔다. Salmonella typhimurium , Salmonella typhi : Salmonella Typhimurium and Salmonella typhi specifically reacted with various Salmonella strains. As a result, various patterns of microarray experiments were observed according to Salmonella serovar .

장내 미생물에 해당하는 식중독균들에 대하여 실험을 수행한 결과, Salmonella typhimurium에 특이적인 프로브들은 Salmonella spp.에서 반응하는 것으로 확인할 수 있었으며, Salmonella typhi 에 특이적인 프로브들은 인간에 특이적으로 질병을 일으키는 것으로 알려져 있는 Salmonella typhi 및 paratyphi A에 특이적인 것을 확인할 수 있었다. Results of the experiment with respect to the corresponding to the intestinal microflora sikjungdokgyun, specific probes for Salmonella typhimurium have been able to check that the reaction in Salmonella spp., Salmonella Probes specific to typhi are Salmonella known to cause disease in humans Specific to typhi and paratyphi A were confirmed.

E. coli O157:H7에 특이적인 프로브들도 식중독균인 E. coli O157:H7에만 특이적인 것이 확인되었으며, 제작되어진 Yersinia enterocolitica의 프로브들도 특이적으로 반응하는 것으로 확인되었다. E. coli O157: H7 on the specific probe is also a sikjungdokgyun E. coli O157: H7 was found to only specific, ready-made Yersinia Probes of enterocolitica were also found to react specifically.

음성 대조군 시료 DNA로 다양한 장내미생물들과 반응시킨 결과 제작된 DNA 칩은 비병원성 장내미생물 (비식중독균)에는 반응하지 않는 것을 확인하였다.As a result of the reaction with various enteric microorganisms with the negative control sample DNA, the produced DNA chip did not react with the non-pathogenic enteric microorganisms (non-food poisoning bacteria).

실시예Example 3:  3: 바실랄레스(Bacillales)에In the Bacillales 속하는 식중독균의  Belonging to food poisoning bacteria 마이크로어레이Microarray 결과 result

Bacillales에 속하는 식중독균들에 대한 결과는 도 4와 같다. Staphylococcus aureus ATCC 25923는 도 4의 A 및 도 7와 같이 특이적인 결과를 나타내었다. Staphylococcus haemolyticus ATCC 29970와 Staphylococcus epidermis ATCC 14990도 FBP-1과 반응한 결과, 도 4 (B, C)와 같이 반응하지 않는 결과를 나타냄으로서 Staphylococcus aureus에서 제작된 10개의 프로브들은 Staphylococcus aureus 와 특이적으로 반응하는 것을 확인할 수 있었다. Listeria monocytogenes의 경우, 도 4 (D-G)와 도 7과 같이 총 7개의 Listeria monocytogenes 의 균주가 프로브들과 특이적으로 반응하는 것을 확인할 수 있었으며, 7개의 균주들의 반응 패턴 이 각각의 균주에 따라서 다른 것을 확인할 수 있었다. 이 결과는 L. monocytogenes 균주들 사이에서도 유전적인 형질이 다르다는 것을 의미한다. 일반적으로 동물에 대하여 병원성 균으로 알려졌으며 게놈 DNA 서열이 L. monocytogenes와 거의 비슷하다고 알려진 Listeria ivanovii도 도 4 와 도 7과 같이 10개의 프로브들과 반응하는 것을 확인할 수 있었다. Bacillus cereus 에 특이적으로 제작된 10개의 프로브들은 도 4 (H-K)와 같이 B. cereus ATCC 10778 및 ATCC 14579 뿐만 아니라 Bacillus anthracis ATCC 14578 와 Bacillus thuringiensis ATCC 10792 와도 반응하는 것을 확인하였다. 이 세 개의 균주들은 병원성 균인 Bacillus cereus 그룹으로 간주되고 있으며, 음성 대조군 균주로 반응했던 비병원성 균인 Bacillus subtilis와는 반응하지 않는 것을 확인할 수 있었다.Results for food poisoning bacteria belonging to Bacillales are shown in FIG. Staphylococcus aureus ATCC 25923 showed specific results as shown in A and 7 of FIG. Staphylococcus haemolyticus ATCC 29970 and Staphylococcus epidermis ATCC 14990 Fig-1 and FBP After a reaction, 4 denotes a reaction that does not result, as the (B, C) Staphylococcus The probes 10 are manufactured in aureus was found to react specifically with Staphylococcus aureus. For Listeria monocytogenes, Fig. 4 (DG) and also as seven there was a strain of seven Listeria monocytogenes found that the reaction specifically with the probe, the reaction patterns of the seven strains in accordance with the respective strain others I could confirm it. This result indicates that genetic traits are different among L. monocytogenes strains. Listeria , commonly known as pathogenic bacteria for animals, whose genomic DNA sequence is similar to L. monocytogenes It can be seen that ivanovii also reacts with 10 probes as shown in FIGS. 4 and 7. Bacillus Ten probes specifically designed for cereus are Bacillus as well as B. cereus ATCC 10778 and ATCC 14579 as shown in FIG. 4 (HK). It was confirmed that the reaction with anthracis ATCC 14578 and Bacillus thuringiensis ATCC 10792. These three strains are Bacillus, a pathogenic bacterium. Bacillus, a non-pathogenic bacterium that is considered a cereus group and responded with a negative control strain It did not react with subtilis .

Bacillus cereus : 총 3종의 Bacillus cereus 균주들에 대하여 특이적으로 발색하는 것을 확인하였으며, Bacillus subtilis , Bacillus thuringiensis의 균주들에 대하여는 발색하지 않았으므로 제작된 프로브가 특이적인 것으로 확인하였다. Bacillus cereus : 3 kinds of Bacillus It was confirmed that the specific development of the cereus strains, Bacillus subtilis , Bacillus Since the strains of thuringiensis did not develop, it was confirmed that the produced probes were specific.

Staphylococcus aureus : 보유하고 있는 5종의 Staphylococcus aureus에 대하여 반응하였으며, Stapylococcus haemolyticus, Stapylococcus epidermidis에 대하여는 반응하지 않는 것을 확인하였다. Staphylococcus aureus : 5 species of Staphylococcus Reacted to aureus , Stapylococcus haemolyticus , Stapylococcus It was confirmed that it did not react with epidermidis .

Listeria monocytogenes : 총 7개의 Listeria monocytogenes 균주들과 Listeria ivanovi 등의 균주들과 실험한 결과로 Listeria monocytogenes ATCC 7644 및 ATCC 19113의 경우 10개의 스팟에서 모두 발색을 하였지만, ATCC 19111, ATCC 19114의 경우에는 각각 다른 패턴으로 발색하는 것을 확인하였다. Listeria welshimeri, Listeria inocua , Listeria grayi , Listeria seeligeri 의 대조구 균들은 발색하지 않는 것으로 확인되었다. Listeria monocytogenes : Total 7 Listeria monocytogenes strains and as a result of experiment with the strain of Listeria, such as Listeria ivanovi The monocytogenes ATCC 7644 and ATCC 19113 developed color in all 10 spots, but the ATCC 19111 and ATCC 19114 developed different patterns. Listeria welshimeri, Listeria inocua , Listeria grayi , Listeria The control bacteria of seeligeri did not appear to develop.

Staphylococcus aureus에 특이적으로 제작된 프로브들은 Staphylococcus aureus에만 특이적으로 반응하는 것을 확인할 수 있었다. Staphylococcus Probes specifically designed for aureus are Staphylococcus It was confirmed that the specific reaction only to aureus .

Listeria의 경우는 제작된 10개의 프로브들에 대한 결과 패턴의 차이는 있으나 Listeria monocytogenes에 반응하는 것으로 확인되었으며, 병원성 균으로 알려진 Listeria ivanovi에도 반응하는 것을 확인할 수 있었다.For Listeria is the difference between the result of the pattern for the production of the probe 10, but Listeria It was found to react in monocytogenes, a pathogenic bacterium known as Listeria It was confirmed that the response to ivanovi .

Bacillus cereus의 경우 대부분의 프로브들이 Bacillus cereus , Bacillus thuringiensis, Bacillus anthracis에만 특이적으로 반응하는 것을 확인하였다. 근래 들어 이 세 종의 미생물은 Bacillus cereus 그룹으로 간주되고 있으며 병원성균으로서 게놈 DNA의 유사도가 상당히 높은 것으로 알려져 있다. Bacillus In cereus , most of the probes are Bacillus cereus , Bacillus Only thuringiensis and Bacillus anthracis reacted specifically. These three microorganisms have recently been known as Bacillus It is considered to be a cereus group and it is known that the similarity of genomic DNA is very high as a pathogenic bacterium.

실시예Example 4:  4: 비브리오나시애(Vibrionaceae)에In Vibrionaceae 속하는 식중독균에 대한  For food poisoning bacteria belonging to 마이크로어레이Microarray 결과 result

Vibrio parahaemolyticus ATCC 17802와 ATCC 33844는 FBP-1 과의 반응 결과, 도 5 와 도 7에 나타난 것과 같이 선별된 10개의 프로브와 특이적으로 반응하는 것을 확인할 수 있었다. 병원성 균으로 알려져 있는 Vibrio cholerae ATCC 14547 과 Vibrio vulnificus ATCC 33815는 프로브들과 반응하지 않았다. Vibrio As a result of reaction with FBP-1, parahaemolyticus ATCC 17802 and ATCC 33844 showed specific reaction with 10 selected probes as shown in FIGS. 5 and 7. Vibrio known as pathogenic bacteria cholerae ATCC 14547 and Vibrio The vulnificus ATCC 33815 did not react with the probes.

Vibrio parahemolyticus : 3종의 Vibrio parahemolyticus에 대하여 특이적이었으며 Vibrio cholerae Vibrio vulnificus에 대하여는 반응하지 않는 것을 확 인하였다. Vibrio parahemolyticus : 3 species of Vibrio specific for parahemolyticus and Vibrio cholerae And Vibrio It was confirmed that it did not respond to vulnificus .

실시예Example 5:  5: ClostridiumClostridium perfringensperfringens , , ClostridiumClostridium botulinumbotulinum  And Campylobacter Campylobacter jejuni jejuni of 마이크로어레이Microarray 결과 result

Campylobacter spp.는 병원성 균으로 알려져 있으며 C. jejuni ATCC 33291 및 ATCC 43429를 반응한 결과, 도 6과 같이 특이적인 결과를 나타내었다. Clostridium botulinumClostridium perfringens에서 특이적으로 제작한 각각 10 개의 프로브들도 C. botulinum ATCC 3502 및 C. perfringens ATCC 3624 와 반응한 결과 도 6과 같이 특이적인 결과를 나타내었다. Campylobacter spp. Is known as a pathogenic bacterium and reacted with C. jejuni ATCC 33291 and ATCC 43429, showing specific results as shown in FIG. Clostridium botulinum and Clostridium Each of the 10 probes specifically prepared in perfringens also reacted with C. botulinum ATCC 3502 and C. perfringens ATCC 3624.

* Clostridium botulinum, Clostridium perfringens , Yersinia enterocolitica: 각각의 Clostridium botulinum ATCC 3502, Clostridium perfringens ATCC 3624, Yersinia enterocolitica ATCC 23715 에 대하여 특이적인 것을 확인하였다. * Clostridium botulinum , Clostridium perfringens , Yersinia enterocolitica : Clostridium of each botulinum ATCC 3502, Clostridium perfringens ATCC 3624, Yersinia enterocolitica It was confirmed that it was specific for ATCC 23715.

* Campylobacter jejuni: 총 2종의 Campylobacter jejuni ATCC 43429는 10개의 스팟이 발색하였으나 대조구에서는 그 발색 패턴이 다르게 나타났으므로 추가적인 프로브의 실험 및 Campylobacter jejuni 다양한 균주들과의 결과를 확인해야 한다. * Campylobacter jejuni : 2 types of Campylobacter jejuni ATCC 43429 developed 10 spots, but the control pattern showed different color development patterns, so experiments with additional probes and Campylobacter jejuni You should check the results with the various strains.

* Clostridium perfringens, Clostridium botulinum , Camphylobacter jejuni에 대하여도 역시 특이적으로 반응한 결과를 확인하였다. Clostridium perfringens , Clostridium botulinum , Camphylobacter Jejuni also showed a specific reaction result.

실시예Example 6: 다양한 미생물들의 게놈  6: genome of various microorganisms DNADNA 들과의  With the field 마이크로어레이Microarray 결과 result

식중독균 및 비병원성 균들을 포함하여 총 69개의 미생물들을 FBP-1 칩과 반응을 하였으며 그 결과는 도 7과 같다. 본 발명에 사용된 균주들은 각각의 식중독균에 선별된 프로브들에 대하여 양성 및 음성 대조군 균주로서 FBP-1 칩과 반응하였다. 그 전체적인 결과는 도 7 에 나타나 있으며 본 발명에서 선별된 각 식중독균에 특이적인 프로브들과 제작된 FBP-1 칩이 식중독균을 구분할 수 있는 능력을 보유하였음을 확인하였다.A total of 69 microorganisms, including food poisoning bacteria and non-pathogenic bacteria, were reacted with FBP-1 chips, and the results are shown in FIG. 7. The strains used in the present invention reacted with FBP-1 chips as positive and negative control strains for probes selected for each food poisoning bacterium. The overall results are shown in Figure 7 and confirmed that the probes specific for each food poisoning bacteria selected in the present invention and the produced FBP-1 chip has the ability to distinguish food poisoning bacteria.

현재 일차적으로 제작된 마이크로어레이 칩에 대하여 확보해왔던 10종의 식중독 미생물들과 대조구 미생물들을 이용하여 게놈 DNA를 추출하였고 마이크로어레이 실험을 수행하여 제작된 대다수의 프로브들이 각각의 식중독균들에 특이적인 것을 확인하였다.Genomic DNA was extracted using 10 kinds of food poisoning microorganisms and control microorganisms that have been secured for the primary microarray chips currently manufactured, and the majority of probes produced by microarray experiments were specific to each food poisoning bacterium. Confirmed.

실험을 수행한 식중독균들은 각각의 특이적인 프로브와 반응하는 것을 확인할 수 있었으며 비식중독균들의 경우 본 발명에서 제작한 마이크로어레이 칩 (FBP-1)과 반응하지 않는 결과를 나타내었다. 본 dendrogram은 본 발명에서 제작된 마이크로어레이 칩 식중독균 및 병원성 균들에 특이적으로 반응함을 보여주었다.It was confirmed that the food poisoning bacteria performed the experiment reacted with each specific probe, and the non-food poisoning bacteria showed a result of not reacting with the microarray chip (FBP-1) manufactured in the present invention. This dendrogram showed the specific response to the microarray chip food poisoning bacteria and pathogenic bacteria produced in the present invention.

도 1은 게놈 서열 및 비교 유전체학을 이용하여 11가지 식중독균으로부터 70-mer 올리고뉴클레오티드 프로브 선발의 작업 흐름도를 보여준다.1 shows a flow chart of the selection of 70-mer oligonucleotide probes from 11 food poisoning bacteria using genomic sequence and comparative genomics.

도 2는 Syto 61 테스트 후의 FBP-1 마이크로어레이의 레이아웃을 보여준다. 각 슬라이드는 8개의 블록으로 이루어져 있으며, 각 블록은 110개 프로브, 양성 대조군 및 음성 대조군으로 이중 스팟팅되어 있다.Figure 2 shows the layout of the FBP-1 microarray after Syto 61 test. Each slide consists of eight blocks, each block double spotted with 110 probes, a positive control and a negative control.

도 3은 Enterobacteriae 식중독균의 마이크로어레이 결과이다.3 is a microarray result of Enterobacteriae food poisoning bacteria.

A, Salmonella enterica serovar Typhimurium ATCC 19585; B, serovar Typhimurium ATCC 13311; C, serovar Typhimurium ATCC 14028; D, Salmonella enterica serovar Typhi ATCC 33459; E, Escherichia coli O157:H7 ATCC 43894; F, E. coli ATCC 11775; G, Yersinia enterocolitica ATCC 23715.A, Salmonella enterica serovar Typhimurium ATCC 19585; B, serovar Typhimurium ATCC 13311; C, serovar Typhimurium ATCC 14028; D, Salmonella enterica serovar Typhi ATCC 33459; E, Escherichia coli 0157: H7 ATCC 43894; F, E. coli ATCC 11775; G, Yersinia enterocolitica ATCC 23715.

도 4는 Bacillales 식중독균의 마이크로어레이 결과이다.4 is a microarray result of Bacillales food poisoning bacteria.

A, Staphylococcus aureus ATCC 25923; B, S. haemolyticus ATCC 29970; C, S. epidermis ATCC 14990; D, Listeria monocytogenes ATCC 19115; E, L. monocytogenes ATCC 19118; F, L. ivanovii ATCC 19119; G, L. inoccua ATCC 33090; H, Bacillus cereus ATCC 10778; I, B. cereus ATCC 14579; J, B. thuringensis ATCC 10792; K, B. antharacis ATCC 14578.A, Staphylococcus aureus ATCC 25923; B, S. haemolyticus ATCC 29970; C, S. epidermis ATCC 14990; D, Listeria monocytogenes ATCC 19115; E, L. monocytogenes ATCC 19118; F, L. ivanovii ATCC 19119; G, L. inoccua ATCC 33090; H, Bacillus cereus ATCC 10778; I, B. cereus ATCC 14579; J, B. thuringensis ATCC 10792; K, B. antharacis ATCC 14578 .

도 5는 Vibrionaceae 식중독균의 마이크로어레이 결과이다.5 is a microarray result of Vibrionaceae food poisoning bacteria.

A, Vibrio parahaemolyticus ATCC 17802; B, V. parahaemolyticus ATCC 33844; C, V. cholerae ATCC 14547; D, V. vulnificus ATCC 33815.A, Vibrio parahaemolyticus ATCC 17802; B, V. parahaemolyticus ATCC 33844; C, V. cholerae ATCC 14547; D, V. vulnificus ATCC 33815.

도 6은 Clostridium perfringens, Clostridium botulinumCampylobacter jejuni의 마이크로어레이 결과이다.6 is Clostridium perfringens , Clostridium Microarray results of botulinum and Campylobacter jejuni .

A, Clostridium perfringens ATCC 3624; B, Clostridium botulinum ATCC 3501; C, Camphylobacter jejuni ATCC 43429; D, C. jejuni ATCC 33291.A, Clostridium perfringens ATCC 3624; B, Clostridium botulinum ATCC 3501; C, Camphylobacter jejuni ATCC 43429; D, C. jejuni ATCC 33291.

도 7은 Genespring 프로그램을 이용한 다양한 박테리아 게놈 DNA의 마이크로어레이 데이터 세트를 이용하여 분석된 계통수(dendrogram)이다.7 is a dendrogram analyzed using microarray data sets of various bacterial genomic DNAs using the Genespring program.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Oligonucleotide probe set for dectecting food-borne pathogens and microarray containing the probe set <130> PN08106 <160> 116 <170> KopatentIn 1.71 <210> 1 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC1312 probe <400> 1 tacacttcgc gttgtggcag aggttattaa gaagcgtgat gataaaaaaa tcattacgct 60 gcaaacaaac 70 <210> 2 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC1501 probe <400> 2 ttgcatgggg atcgcatatt acgattatgt tctctatatg tagggatgtt acaagcagag 60 ctgctagtca 70 <210> 3 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC1801 probe <400> 3 tttatcgtag tgaaaaatca cgtagttctc atactggtgg gaaaggaatg gggcttgcaa 60 tttcgaaaag 70 <210> 4 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC2098 probe <400> 4 tgaggagttt ttttcctcaa cgttatatgc acgtacgaaa caagattggg catatgcacc 60 aaacatgtat 70 <210> 5 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC2140 probe <400> 5 ctccctttac atccagtccc ttttaacgga gaagtcatct cctctcaaac aggagaatat 60 cgaattcgta 70 <210> 6 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC2393 probe <400> 6 aatggtggca cctcaaataa gacaccgatt tgactctgta aaaaaatttg cagaggagga 60 atctattccg 70 <210> 7 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC3742 probe <400> 7 ttgctgagaa acaattaact gctatccatg gtataggacc atggactgcc aactatgttt 60 taatgcgttg 70 <210> 8 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC4039 probe <400> 8 gatgtttcag cacgtcgagg ttataaagca catgatgtaa tctcactttc aagtagcaca 60 cctaaccttg 70 <210> 9 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC4292 probe <400> 9 aaaattgtgc aagttggagc ggaatttcat gtcggaattt tgtttgtcgc atcatatgct 60 catcctttaa 70 <210> 10 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC4598 probe <400> 10 tgtatttgta ggaggtattc tcttagtgct tcctgggtat gtgacagata tgctagggtt 60 tatctgcgtg 70 <210> 11 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo0754 probe <400> 11 ggaacttaat tctgacgcca aattagtgac acgagtagaa cgaatcgatg gcaaatggta 60 cattaatgca 70 <210> 12 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo2443 probe <400> 12 tgcatttttt ggaatagcag taatttgttt gttattccct tggatgtcta gcgggataga 60 tgcagcaaca 70 <210> 13 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo2732 probe <400> 13 attcgtttag gaattgagca acttccaaaa aaagaaattg agcaagttgc gacaagaatt 60 gcttatgcgc 70 <210> 14 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo1030 probe <400> 14 gcaaccaaca gcaggcctta ttttacttgg cactaattta aacgctgaag aaattaaccg 60 aatacagcaa 70 <210> 15 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo1033 probe <400> 15 gttagttatg gtgcacttgg tatgagccat cattcgcttc aagatattgc tgttactaga 60 gcgattccta 70 <210> 16 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo1036 probe <400> 16 gtcagttaga gccaatcaag ccagttatca attcagaacc gtgttacgaa ttgatgggat 60 atagtcgaca 70 <210> 17 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo2821 probe <400> 17 gacatatgtg tatactaaaa acatcgaagc agcagagccg ataacagtga attacgtgga 60 tgctaatggc 70 <210> 18 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo0017 probe <400> 18 ctaagccaaa aaagaaaacc gtcgctgctg ttaaaaagaa aaaagagaca cctccaaaac 60 caaaagaacc 70 <210> 19 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo0257 probe <400> 19 aagataaaat tgtaccgaat ttggttggag tggacatcgg ttgtgggttg tatgtagtga 60 agctaaaacc 70 <210> 20 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo0459 probe <400> 20 gcagtttaaa gtagccctat ataaggcttg caccgaggag tctaagaaat cagagagtgc 60 gttcattaaa 70 <210> 21 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0020 probe <400> 21 agaaggctct aatcaaattc aagtcacttg gggtgaaaaa ggcgtctttg attatcgtcg 60 ttcgttattg 70 <210> 22 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0021 probe <400> 22 agcccacaga tggtgcagat aagaagcatc aagttattgg agtgagaaaa gcaatcgagg 60 cattatatta 70 <210> 23 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0089 probe <400> 23 acaatcaatt gcagaagcac gagcacttct agatgaattt gaagcccaaa tggacgaaga 60 cgttaaaata 70 <210> 24 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0101 probe <400> 24 atccgtactt agcgttggac aatcaaatga atatgcgtgc aacggaaatg atccatttga 60 acattaatgc 70 <210> 25 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0110 probe <400> 25 ggtccagaac tagcagacat tattccatca ctagcaacaa tggtggtgtt agcatttgtt 60 tgtcgtaaat 70 <210> 26 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0149 probe <400> 26 tatctaaagc agacggtaca gcagttaaag tcgcaccaaa aactgtagtg aatctaatcg 60 gtgcattctt 70 <210> 27 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0155 probe <400> 27 ttccctggtg tccttattcg aacttccaca gaaagaccgg aagtactaga taaaggtacg 60 gttattgtag 70 <210> 28 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0178 probe <400> 28 tttggttacc tcaagaccga aatcaagtaa agcaaagtga tgacaagagg catgcaagtc 60 gttatcaaaa 70 <210> 29 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0182 probe <400> 29 atgattccca aagtgctaga tgccaaaaat gcgattgaaa atggctgtcc taaagttatc 60 attgcatcag 70 <210> 30 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0190 probe <400> 30 cgttgataat ttgaataacg gacgttatca aggcgaaatg caaattgtga gacaaacgct 60 tagtgcacat 70 <210> 31 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0001 probe <400> 31 ggcggtatac tttgtgggaa tctaaacaca ttctcagaat cgtatttgac cttaacttgg 60 gccagtagat 70 <210> 32 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0002 probe <400> 32 acgatgttat agatatcctc cggtactccg atatttttac aggctttcat attaacgggc 60 aaggtagcca 70 <210> 33 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0004 probe <400> 33 aattgaacgg cgagaatatg gagttgcaca caataaggca tccgcttttg gatatttaag 60 ttctgtcatt 70 <210> 34 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0007 probe <400> 34 tctgttatct tgaaaacctt accagataca ctgctatgaa ttctagcccc caatccatta 60 ggctctgcta 70 <210> 35 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0008 probe <400> 35 tggaagccct catctgtatc tttatccact atatctgcat caccatttgt gtttatgcga 60 gcttcttctc 70 <210> 36 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0009 probe <400> 36 cgcagctgtt tggataggta atgacaattc ttctacagtg gatggggtat atagtagtac 60 tgcagctaga 70 <210> 37 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0011 probe <400> 37 gaacagttag ctgtacctcc tctcatttct ggtccataag atggtaacca tgaaacttcc 60 atatttgcgt 70 <210> 38 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0012 probe <400> 38 ctcgtgatat atccttccaa tctaagttga tcataggcat tttctaccga gtttctgccg 60 acctgcaatt 70 <210> 39 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0013 probe <400> 39 acccgctgca aaacaattaa aggagcgatt tggaactcca tatttattgg ctagacccta 60 tggaattaag 70 <210> 40 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0017 probe <400> 40 aaagcctgct agtcccatta caacattaag tcgggttact cctaatgcat gatcatgata 60 ccacaaagtt 70 <210> 41 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE0192 probe <400> 41 ggtggtctta ctactcttgg agctaatact ttttcaatgg ctgtagttgg gcctttagta 60 tcatacggac 70 <210> 42 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE0590 probe <400> 42 cttagttttc tttggaacag ttgtaactca cttttttgga ggatcagcag gtagggaagg 60 aactggagta 70 <210> 43 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE0638 probe <400> 43 caactaatat agccttagaa atggggtatg catttggaac tcaccaagaa atgctatttg 60 caacaggggt 70 <210> 44 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE1030 probe <400> 44 agttgggctt acggaaactt atttgatact tacgttttag atagcgaccc agcagataaa 60 tgggatgctt 70 <210> 45 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE1094 probe <400> 45 catttactgg agctatgtac acattaggaa tgccacctga acttatggga atgggtagag 60 cattaaacga 70 <210> 46 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE2193 probe <400> 46 cttgcacctc ctactcaaga tttaagtgtt gcagtggcat tagcaattac ttcctttgtt 60 gttgttcatg 70 <210> 47 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE2318 probe <400> 47 atgggaaatc aagtatgtcc acttccaaca gcagtattgt caacacatac agatggattt 60 ggaaaaccag 70 <210> 48 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE2505 probe <400> 48 gtatgactgc atattcaata gcctatgcta gaataggaac aaacgctgtt gctagcatgc 60 agattgctac 70 <210> 49 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE2577 probe <400> 49 gaggtagacg gggctagaag cttaatagaa aaagcgtcaa tgcaattagg caaagctcat 60 aaatcacaaa 70 <210> 50 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE2609 probe <400> 50 atctactagg aggagcaatt ggtgttgtta taacctatac tgttatggct gctgtaggaa 60 gcttaggacc 70 <210> 51 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj0150c probe <400> 51 ttccaaatta tgttttcgca gaagtaaatg cgataaaaat ggcagcaaga cgtgctggag 60 aggatatcat 70 <210> 52 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj0933c probe <400> 52 tccacttatt cctttttctc ctatgccagg aggagcttta acggcaaata cacaaatgat 60 gagggataat 70 <210> 53 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj1170c probe <400> 53 gtgggcctgt actttatgta gatagagctt atcttgacta ctatatcaca cctgagtgga 60 ttgcaaccgt 70 <210> 54 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj0921c probe <400> 54 ggagtggctc aagctgcaac tacaaaaaaa gctataggtg aagctgctaa aaaaattggc 60 attgatgtta 70 <210> 55 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj0998c probe <400> 55 taaaggattt ggcaagttat cttaaaggcg ctaaagctac cataaagcca agcaatgctt 60 ttatgggtga 70 <210> 56 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj1134 probe <400> 56 aaggcggcat aagacaaatg ctaagcgctc taaaaaaagg gagaactttg ggaattttaa 60 ctgatcaaga 70 <210> 57 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj1444c probe <400> 57 aaagagcttg aaactctagc tttaactagt ggttctgtta cagctcaagg tgcagctata 60 agagctcaac 70 <210> 58 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj0116 probe <400> 58 tttgctttag ggcattcttt gggagaattt tcagccttag ctgtcaatgg tgcttttgat 60 tttttagagg 70 <210> 59 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj0190c probe <400> 59 aaatcactat caatcttagt ccttcaggca tcccaaaaaa gggatcgcat tttgatttgg 60 ctatcgcttt 70 <210> 60 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj1347c probe <400> 60 atcagggtat gtttgcactt ttttggctta tcattatcgt tgcagcttgt gatagtgggg 60 cgtattttat 70 <210> 61 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP0434 probe <400> 61 gataccttgg gtaaggatta cactaaactc taccaacata tggcgaagac ctcttcagaa 60 cttctgccta 70 <210> 62 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP0626 probe <400> 62 agagaccgtc cattcgggat tccaaactac gttctattct atgagaaaag gtctaactcg 60 ttttcttcgt 70 <210> 63 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP0656 probe <400> 63 tagaacggtt gccttgagcg atcttagtcg aattcaactt gaactagaga catcttatga 60 tggtgggtat 70 <210> 64 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP0753 probe <400> 64 acatgattat cggctccgta catgtcggtg ataaaagcat gtatccactt ccgaaaaaca 60 tcactaagtt 70 <210> 65 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP0948 probe <400> 65 tactgatttc cgattcaggt aacgacttat atctacctaa gcaagttcgt gcaaccgtga 60 cattgtatgc 70 <210> 66 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP0953 probe <400> 66 ataacttaac tggattgagg aatcgtcgct ctttgcaagt taagtatcgc caaggggtga 60 ataaggattt 70 <210> 67 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP1284 probe <400> 67 ccgctgtact acagctacgc agaaaactcg aagttaaacg ataagtacac cgagataatc 60 aaagagcaaa 70 <210> 68 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP1365 probe <400> 68 cagatgttta cacgattacg gcaattcagt tggagaatac aacagagttt gaaggtttcc 60 atggtgtgaa 70 <210> 69 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP1386 probe <400> 69 gtatggtgca ttgcttactg catactcttc ttactcttat gacatgatcc aaaggtacgg 60 tcaatccgaa 70 <210> 70 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP1517 probe <400> 70 tcggtcgatt agtcgagcag caatggtcag gtagagaaaa aaaatacata tacaatgcac 60 agaaccgaat 70 <210> 71 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0005 probe <400> 71 gatattgatt actgtgctta atatcaggat agagcatacg ggccagtttt agcgtgcaaa 60 tccattgacc 70 <210> 72 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0007 probe <400> 72 gctcacccga cttcatcagc attatggcta tctctgtgaa cagatcgaag aaatcgaaag 60 agagttaaaa 70 <210> 73 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0009 probe <400> 73 tttagttcgt tcagcgctgg tgcctgatag tcttccggaa catattgtta atactatcat 60 gtctgcattt 70 <210> 74 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0011 probe <400> 74 aaatacagga atgagacacg gcgagatatg cgcattaagt tgggaagata tcgacactaa 60 aaattggact 70 <210> 75 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0013 probe <400> 75 ttttaactct ctttcgattt cttcgatctg ttcacagaga tagccataat gctgatgaag 60 tcgggtgagc 70 <210> 76 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0014 probe <400> 76 ttgcagatca aagacattct ttcagagaac ccggcagtct tcagtatttc ggtgccataa 60 tacataatca 70 <210> 77 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0015 probe <400> 77 gattgtcttg ccaactaatt gcttcaattg acaagatcgg ttgtagtgag ttttcatttg 60 gcgtgtatcc 70 <210> 78 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0016 probe <400> 78 tactcaaaaa aagaagtacc aaaaacctga ttaaaagcac aatgaccacg cacactgtcg 60 gcaactaaac 70 <210> 79 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0017 probe <400> 79 tcttcaacct gccttcacct tgcaagatcg agtttttgag gtttgtcagc agtctgatat 60 taatcttact 70 <210> 80 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0020 probe <400> 80 tgatattgga aatgttgttc acccaacata aaagggagat tcagtgaagc ggtcaaatta 60 atcagtcggg 70 <210> 81 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z0146 probe <400> 81 agattgcgtc atggtatata ggtaccaatg accagccgtg gatcaacttt tacattgaca 60 atgactcttt 70 <210> 82 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z0152 probe <400> 82 ctgaagttca ggaactgaca ttagacgcag aaggtaaagg ccagtacgta tttaacgcat 60 cttacgttaa 70 <210> 83 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z0372 probe <400> 83 caataggtgg agtgacattt attcgtcgtc caacttttaa cgatcgcttc atcgtggaag 60 atggaaatac 70 <210> 84 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z0502 probe <400> 84 gatttcttca tccggatacg tatctgccga taaactgcac aaactctata agacaaattt 60 ggaagacgcg 70 <210> 85 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z1019 probe <400> 85 gtactgatac atacaaacag taaaagatct gttaacgcaa ttaactgatg gcatggagct 60 cttcgcgctg 70 <210> 86 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z1139 probe <400> 86 tgcattctgg ttctgtcagc actgataata tccttttggc tggatgttga ctctgttact 60 cgagtattac 70 <210> 87 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z1140 probe <400> 87 tatgttttta gttgtcgggg agacagcaag aagtcaaaac tatgctctga atggatattc 60 tcgtggtacc 70 <210> 88 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z1535 probe <400> 88 taggtcaaac tgaagatggt cggacagcgc ttattgagtt cggaaaaatt aatatgaccg 60 acacctattt 70 <210> 89 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z1543 probe <400> 89 gcgatcgttg gagtattcgc gggtttgaaa atagcgtagg tttgtcaggg aatgatggtt 60 tttatataaa 70 <210> 90 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z1554 probe <400> 90 tttataaaaa ccagtaaccc ggaattgatt tgggtgacgg tggtattacc tattttgaca 60 gcaacaggtg 70 <210> 91 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY0201 probe <400> 91 tgaaaagtag ttcgagcgcc cagatggtac tcaaacctaa cgactcaagc tcaatttata 60 aagataccac 70 <210> 92 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY0322 probe <400> 92 acgatgttga tactatcggt tttcagggga aagggctatt tttttctgac agactgatcg 60 atgcaataca 70 <210> 93 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY1076 probe <400> 93 attttttaaa tatgtcgacc acatcatttt ccaataacag tcaggagtta gcagcccaca 60 gttcaccagg 70 <210> 94 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY1607 probe <400> 94 gcatttctta agagtgcgtt tgaacactta catgcactaa gtaaggctgg tgaaccactt 60 agtcttgaaa 70 <210> 95 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY2020 probe <400> 95 ataattgttt tctcgaatta gccagagaat cattgcagca caatggtgag caatggacac 60 gtaatgctat 70 <210> 96 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY3673 probe <400> 96 ttatgtaaat tatcaggagt actaagccaa gcctgtcaat cgacgctgat tcctcaggat 60 gagtttgaac 70 <210> 97 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY3922 probe <400> 97 gaaatgtacc ggaatgttgg atgagaatat cgtctattcg acatttaatg ctgatcccgt 60 tgattccagc 70 <210> 98 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY0314 probe <400> 98 aagcggttgt atctgaattt aatttgtcag atataaatcg cggtggaatg acgaaagcac 60 aggcagaaaa 70 <210> 99 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY1891 probe <400> 99 aaatatctgg acctatccgc tttttgttaa taccttttca gcaaatgctc ttgtgggact 60 atcatcgtgc 70 <210> 100 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY2356 probe <400> 100 aaataaaacg ggtgattttt ataagcccag gcaggcttat ggtgatttgg catcggtaaa 60 catggttata 70 <210> 101 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM0159 probe <400> 101 ccggtgagca cagaatattg aaaactgact ttgcattact ttgcccaaat tgtcacaaag 60 ctgttcatat 70 <210> 102 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM2743 probe <400> 102 tgacggagtt cgctttgagg acttgttttc aaaaataatg tactataagt cgccagattt 60 tcagcaggtg 70 <210> 103 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM2747 probe <400> 103 gaatccatta tgattcgccg tgtagtggta aaccagaagt aataaatgct gtgcaattat 60 tgcgctctca 70 <210> 104 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM2754 probe <400> 104 tgatttaata ggcaaaaaaa gcgaagaata tgcattaaac tgggaaaaaa accgcctaat 60 cggtctcggc 70 <210> 105 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM2761 probe <400> 105 taagcaagat tgagctgtta gagttagttc gacgtggacg tataaaattc gtcgcattcc 60 aaaatctcca 70 <210> 106 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM2762 probe <400> 106 cgtagctaat acgaaaaaag aaacagaaaa aatcggagcg acgataaagg tagtgctagg 60 cgttttcgtt 70 <210> 107 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM0654 probe <400> 107 ttattacatc ggaaaaggta tcaaacaaga ttaccagcag gcaatatact ggtttcgaaa 60 agcggcagac 70 <210> 108 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM0762 probe <400> 108 tatgcaaaaa aagaacaact tgcatgctgc catgataccg gcacttgtat cgttataatg 60 gaaattggac 70 <210> 109 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM0763 probe <400> 109 gcatttccga gagatttatc cagagatgga actctctctt gttgaagagg ggacttttgg 60 tttacatgat 70 <210> 110 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM0764 probe <400> 110 aaacaaccca gtcttgtttt ttcagcaacg catgctttgt ctttctcttt tgtaccgcat 60 ttgttaaaac 70 <210> 111 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> tubulin bete-4 chain probe <400> 111 aagtccagtg tctgtgatat tgcaccaaag ggtttgaaaa tggcgtctac tttcattggt 60 aactcaacct 70 <210> 112 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> actin 3 probe <400> 112 aggttcttta ccagccatct atgattggta tggagaatgc tggtatccat gaaaccacct 60 ataactccat 70 <210> 113 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Actin3-f primer <400> 113 ctcttgacta cgagcaggaa 20 <210> 114 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Actin3-r primer <400> 114 agtccttcct gatgtcgaca 20 <210> 115 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tb4-f primer <400> 115 aagaggttga cgagcagatg 20 <210> 116 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tb4-r primer <400> 116 gcctttctcc tgaacatagc 20 <110> University-Industry Cooperation Group of Kyung Hee University <120> Oligonucleotide probe set for dectecting food-borne pathogens and          microarray containing the probe set <130> PN08106 <160> 116 <170> KopatentIn 1.71 <210> 1 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC1312 probe <400> 1 tacacttcgc gttgtggcag aggttattaa gaagcgtgat gataaaaaaa tcattacgct 60 gcaaacaaac 70 <210> 2 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC1501 probe <400> 2 ttgcatgggg atcgcatatt acgattatgt tctctatatg tagggatgtt acaagcagag 60 ctgctagtca 70 <210> 3 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC1801 probe <400> 3 tttatcgtag tgaaaaatca cgtagttctc atactggtgg gaaaggaatg gggcttgcaa 60 tttcgaaaag 70 <210> 4 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC2098 probe <400> 4 tgaggagttt ttttcctcaa cgttatatgc acgtacgaaa caagattggg catatgcacc 60 aaacatgtat 70 <210> 5 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC2140 probe <400> 5 ctccctttac atccagtccc ttttaacgga gaagtcatct cctctcaaac aggagaatat 60 cgaattcgta 70 <210> 6 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC2393 probe <400> 6 aatggtggca cctcaaataa gacaccgatt tgactctgta aaaaaatttg cagaggagga 60 atctattccg 70 <210> 7 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC3742 probe <400> 7 ttgctgagaa acaattaact gctatccatg gtataggacc atggactgcc aactatgttt 60 taatgcgttg 70 <210> 8 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC4039 probe <400> 8 gatgtttcag cacgtcgagg ttataaagca catgatgtaa tctcactttc aagtagcaca 60 cctaaccttg 70 <210> 9 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC4292 probe <400> 9 aaaattgtgc aagttggagc ggaatttcat gtcggaattt tgtttgtcgc atcatatgct 60 catcctttaa 70 <210> 10 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> BC4598 probe <400> 10 tgtatttgta ggaggtattc tcttagtgct tcctgggtat gtgacagata tgctagggtt 60 tatctgcgtg 70 <210> 11 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo0754 probe <400> 11 ggaacttaat tctgacgcca aattagtgac acgagtagaa cgaatcgatg gcaaatggta 60 cattaatgca 70 <210> 12 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo2443 probe <400> 12 tgcatttttt ggaatagcag taatttgttt gttattccct tggatgtcta gcgggataga 60 tgcagcaaca 70 <210> 13 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo2732 probe <400> 13 attcgtttag gaattgagca acttccaaaa aaagaaattg agcaagttgc gacaagaatt 60 gcttatgcgc 70 <210> 14 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo1030 probe <400> 14 gcaaccaaca gcaggcctta ttttacttgg cactaattta aacgctgaag aaattaaccg 60 aatacagcaa 70 <210> 15 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo1033 probe <400> 15 gttagttatg gtgcacttgg tatgagccat cattcgcttc aagatattgc tgttactaga 60 gcgattccta 70 <210> 16 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo1036 probe <400> 16 gtcagttaga gccaatcaag ccagttatca attcagaacc gtgttacgaa ttgatgggat 60 atagtcgaca 70 <210> 17 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo2821 probe <400> 17 gacatatgtg tatactaaaa acatcgaagc agcagagccg ataacagtga attacgtgga 60 tgctaatggc 70 <210> 18 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo0017 probe <400> 18 ctaagccaaa aaagaaaacc gtcgctgctg ttaaaaagaa aaaagagaca cctccaaaac 60 caaaagaacc 70 <210> 19 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo0257 probe <400> 19 aagataaaat tgtaccgaat ttggttggag tggacatcgg ttgtgggttg tatgtagtga 60 agctaaaacc 70 <210> 20 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> lmo0459 probe <400> 20 gcagtttaaa gtagccctat ataaggcttg caccgaggag tctaagaaat cagagagtgc 60 gttcattaaa 70 <210> 21 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0020 probe <400> 21 agaaggctct aatcaaattc aagtcacttg gggtgaaaaa ggcgtctttg attatcgtcg 60 ttcgttattg 70 <210> 22 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0021 probe <400> 22 agcccacaga tggtgcagat aagaagcatc aagttattgg agtgagaaaa gcaatcgagg 60 cattatatta 70 <210> 23 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0089 probe <400> 23 acaatcaatt gcagaagcac gagcacttct agatgaattt gaagcccaaa tggacgaaga 60 cgttaaaata 70 <210> 24 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0101 probe <400> 24 atccgtactt agcgttggac aatcaaatga atatgcgtgc aacggaaatg atccatttga 60 acattaatgc 70 <210> 25 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0110 probe <400> 25 ggtccagaac tagcagacat tattccatca ctagcaacaa tggtggtgtt agcatttgtt 60 tgtcgtaaat 70 <210> 26 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0149 probe <400> 26 tatctaaagc agacggtaca gcagttaaag tcgcaccaaa aactgtagtg aatctaatcg 60 gtgcattctt 70 <210> 27 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0155 probe <400> 27 ttccctggtg tccttattcg aacttccaca gaaagaccgg aagtactaga taaaggtacg 60 gttattgtag 70 <210> 28 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0178 probe <400> 28 tttggttacc tcaagaccga aatcaagtaa agcaaagtga tgacaagagg catgcaagtc 60 gttatcaaaa 70 <210> 29 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0182 probe <400> 29 atgattccca aagtgctaga tgccaaaaat gcgattgaaa atggctgtcc taaagttatc 60 attgcatcag 70 <210> 30 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> SAV0190 probe <400> 30 cgttgataat ttgaataacg gacgttatca aggcgaaatg caaattgtga gacaaacgct 60 tagtgcacat 70 <210> 31 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0001 probe <400> 31 ggcggtatac tttgtgggaa tctaaacaca ttctcagaat cgtatttgac cttaacttgg 60 gccagtagat 70 <210> 32 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0002 probe <400> 32 acgatgttat agatatcctc cggtactccg atatttttac aggctttcat attaacgggc 60 aaggtagcca 70 <210> 33 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0004 probe <400> 33 aattgaacgg cgagaatatg gagttgcaca caataaggca tccgcttttg gatatttaag 60 ttctgtcatt 70 <210> 34 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0007 probe <400> 34 tctgttatct tgaaaacctt accagataca ctgctatgaa ttctagcccc caatccatta 60 ggctctgcta 70 <210> 35 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0008 probe <400> 35 tggaagccct catctgtatc tttatccact atatctgcat caccatttgt gtttatgcga 60 gcttcttctc 70 <210> 36 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0009 probe <400> 36 cgcagctgtt tggataggta atgacaattc ttctacagtg gatggggtat atagtagtac 60 tgcagctaga 70 <210> 37 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0011 probe <400> 37 gaacagttag ctgtacctcc tctcatttct ggtccataag atggtaacca tgaaacttcc 60 atatttgcgt 70 <210> 38 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0012 probe <400> 38 ctcgtgatat atccttccaa tctaagttga tcataggcat tttctaccga gtttctgccg 60 acctgcaatt 70 <210> 39 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0013 probe <400> 39 acccgctgca aaacaattaa aggagcgatt tggaactcca tatttattgg ctagacccta 60 tggaattaag 70 <210> 40 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CB0017 probe <400> 40 aaagcctgct agtcccatta caacattaag tcgggttact cctaatgcat gatcatgata 60 ccacaaagtt 70 <210> 41 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE0192 probe <400> 41 ggtggtctta ctactcttgg agctaatact ttttcaatgg ctgtagttgg gcctttagta 60 tcatacggac 70 <210> 42 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE0590 probe <400> 42 cttagttttc tttggaacag ttgtaactca cttttttgga ggatcagcag gtagggaagg 60 aactggagta 70 <210> 43 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE0638 probe <400> 43 caactaatat agccttagaa atggggtatg catttggaac tcaccaagaa atgctatttg 60 caacaggggt 70 <210> 44 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE1030 probe <400> 44 agttgggctt acggaaactt atttgatact tacgttttag atagcgaccc agcagataaa 60 tgggatgctt 70 <210> 45 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE1094 probe <400> 45 catttactgg agctatgtac acattaggaa tgccacctga acttatggga atgggtagag 60 cattaaacga 70 <210> 46 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE2193 probe <400> 46 cttgcacctc ctactcaaga tttaagtgtt gcagtggcat tagcaattac ttcctttgtt 60 gttgttcatg 70 <210> 47 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE2318 probe <400> 47 atgggaaatc aagtatgtcc acttccaaca gcagtattgt caacacatac agatggattt 60 ggaaaaccag 70 <210> 48 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE2505 probe <400> 48 gtatgactgc atattcaata gcctatgcta gaataggaac aaacgctgtt gctagcatgc 60 agattgctac 70 <210> 49 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE2577 probe <400> 49 gaggtagacg gggctagaag cttaatagaa aaagcgtcaa tgcaattagg caaagctcat 60 aaatcacaaa 70 <210> 50 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CPE2609 probe <400> 50 atctactagg aggagcaatt ggtgttgtta taacctatac tgttatggct gctgtaggaa 60 gcttaggacc 70 <210> 51 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj0150c probe <400> 51 ttccaaatta tgttttcgca gaagtaaatg cgataaaaat ggcagcaaga cgtgctggag 60 aggatatcat 70 <210> 52 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj0933c probe <400> 52 tccacttatt cctttttctc ctatgccagg aggagcttta acggcaaata cacaaatgat 60 gagggataat 70 <210> 53 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj1170c probe <400> 53 gtgggcctgt actttatgta gatagagctt atcttgacta ctatatcaca cctgagtgga 60 ttgcaaccgt 70 <210> 54 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj0921c probe <400> 54 ggagtggctc aagctgcaac tacaaaaaaa gctataggtg aagctgctaa aaaaattggc 60 attgatgtta 70 <210> 55 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj0998c probe <400> 55 taaaggattt ggcaagttat cttaaaggcg ctaaagctac cataaagcca agcaatgctt 60 ttatgggtga 70 <210> 56 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj1134 probe <400> 56 aaggcggcat aagacaaatg ctaagcgctc taaaaaaagg gagaactttg ggaattttaa 60 ctgatcaaga 70 <210> 57 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj1444c probe <400> 57 aaagagcttg aaactctagc tttaactagt ggttctgtta cagctcaagg tgcagctata 60 agagctcaac 70 <210> 58 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj0116 probe <400> 58 tttgctttag ggcattcttt gggagaattt tcagccttag ctgtcaatgg tgcttttgat 60 tttttagagg 70 <210> 59 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj0190c probe <400> 59 aaatcactat caatcttagt ccttcaggca tcccaaaaaa gggatcgcat tttgatttgg 60 ctatcgcttt 70 <210> 60 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cj1347c probe <400> 60 atcagggtat gtttgcactt ttttggctta tcattatcgt tgcagcttgt gatagtgggg 60 cgtattttat 70 <210> 61 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP0434 probe <400> 61 gataccttgg gtaaggatta cactaaactc taccaacata tggcgaagac ctcttcagaa 60 cttctgccta 70 <210> 62 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP0626 probe <400> 62 agagaccgtc cattcgggat tccaaactac gttctattct atgagaaaag gtctaactcg 60 ttttcttcgt 70 <210> 63 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP0656 probe <400> 63 tagaacggtt gccttgagcg atcttagtcg aattcaactt gaactagaga catcttatga 60 tggtgggtat 70 <210> 64 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP0753 probe <400> 64 acatgattat cggctccgta catgtcggtg ataaaagcat gtatccactt ccgaaaaaca 60 tcactaagtt 70 <210> 65 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP0948 probe <400> 65 tactgatttc cgattcaggt aacgacttat atctacctaa gcaagttcgt gcaaccgtga 60 cattgtatgc 70 <210> 66 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP0953 probe <400> 66 ataacttaac tggattgagg aatcgtcgct ctttgcaagt taagtatcgc caaggggtga 60 ataaggattt 70 <210> 67 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP1284 probe <400> 67 ccgctgtact acagctacgc agaaaactcg aagttaaacg ataagtacac cgagataatc 60 aaagagcaaa 70 <210> 68 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP1365 probe <400> 68 cagatgttta cacgattacg gcaattcagt tggagaatac aacagagttt gaaggtttcc 60 atggtgtgaa 70 <210> 69 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP1386 probe <400> 69 gtatggtgca ttgcttactg catactcttc ttactcttat gacatgatcc aaaggtacgg 60 tcaatccgaa 70 <210> 70 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> VP1517 probe <400> 70 tcggtcgatt agtcgagcag caatggtcag gtagagaaaa aaaatacata tacaatgcac 60 agaaccgaat 70 <210> 71 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0005 probe <400> 71 gatattgatt actgtgctta atatcaggat agagcatacg ggccagtttt agcgtgcaaa 60 tccattgacc 70 <210> 72 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0007 probe <400> 72 gctcacccga cttcatcagc attatggcta tctctgtgaa cagatcgaag aaatcgaaag 60 agagttaaaa 70 <210> 73 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0009 probe <400> 73 tttagttcgt tcagcgctgg tgcctgatag tcttccggaa catattgtta atactatcat 60 gtctgcattt 70 <210> 74 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0011 probe <400> 74 aaatacagga atgagacacg gcgagatatg cgcattaagt tgggaagata tcgacactaa 60 aaattggact 70 <210> 75 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0013 probe <400> 75 ttttaactct ctttcgattt cttcgatctg ttcacagaga tagccataat gctgatgaag 60 tcgggtgagc 70 <210> 76 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0014 probe <400> 76 ttgcagatca aagacattct ttcagagaac ccggcagtct tcagtatttc ggtgccataa 60 tacataatca 70 <210> 77 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0015 probe <400> 77 gattgtcttg ccaactaatt gcttcaattg acaagatcgg ttgtagtgag ttttcatttg 60 gcgtgtatcc 70 <210> 78 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0016 probe <400> 78 tactcaaaaa aagaagtacc aaaaacctga ttaaaagcac aatgaccacg cacactgtcg 60 gcaactaaac 70 <210> 79 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0017 probe <400> 79 tcttcaacct gccttcacct tgcaagatcg agtttttgag gtttgtcagc agtctgatat 60 taatcttact 70 <210> 80 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> YE0020 probe <400> 80 tgatattgga aatgttgttc acccaacata aaagggagat tcagtgaagc ggtcaaatta 60 atcagtcggg 70 <210> 81 <211> 70 <212> DNA <213> Artificial Sequence <220> <0112> Z0146 probe <400> 81 agattgcgtc atggtatata ggtaccaatg accagccgtg gatcaacttt tacattgaca 60 atgactcttt 70 <210> 82 <211> 70 <212> DNA <213> Artificial Sequence <220> <0112> Z0152 probe <400> 82 ctgaagttca ggaactgaca ttagacgcag aaggtaaagg ccagtacgta tttaacgcat 60 cttacgttaa 70 <210> 83 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z0372 probe <400> 83 caataggtgg agtgacattt attcgtcgtc caacttttaa cgatcgcttc atcgtggaag 60 atggaaatac 70 <210> 84 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z0502 probe <400> 84 gatttcttca tccggatacg tatctgccga taaactgcac aaactctata agacaaattt 60 ggaagacgcg 70 <210> 85 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z1019 probe <400> 85 gtactgatac atacaaacag taaaagatct gttaacgcaa ttaactgatg gcatggagct 60 cttcgcgctg 70 <210> 86 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z1139 probe <400> 86 tgcattctgg ttctgtcagc actgataata tccttttggc tggatgttga ctctgttact 60 cgagtattac 70 <210> 87 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z1140 probe <400> 87 tatgttttta gttgtcgggg agacagcaag aagtcaaaac tatgctctga atggatattc 60 tcgtggtacc 70 <210> 88 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z1535 probe <400> 88 taggtcaaac tgaagatggt cggacagcgc ttattgagtt cggaaaaatt aatatgaccg 60 acacctattt 70 <210> 89 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z1543 probe <400> 89 gcgatcgttg gagtattcgc gggtttgaaa atagcgtagg tttgtcaggg aatgatggtt 60 tttatataaa 70 <210> 90 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Z1554 probe <400> 90 tttataaaaa ccagtaaccc ggaattgatt tgggtgacgg tggtattacc tattttgaca 60 gcaacaggtg 70 <210> 91 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY0201 probe <400> 91 tgaaaagtag ttcgagcgcc cagatggtac tcaaacctaa cgactcaagc tcaatttata 60 aagataccac 70 <210> 92 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY0322 probe <400> 92 acgatgttga tactatcggt tttcagggga aagggctatt tttttctgac agactgatcg 60 atgcaataca 70 <210> 93 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY1076 probe <400> 93 attttttaaa tatgtcgacc acatcatttt ccaataacag tcaggagtta gcagcccaca 60 gttcaccagg 70 <210> 94 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY1607 probe <400> 94 gcatttctta agagtgcgtt tgaacactta catgcactaa gtaaggctgg tgaaccactt 60 agtcttgaaa 70 <210> 95 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY2020 probe <400> 95 ataattgttt tctcgaatta gccagagaat cattgcagca caatggtgag caatggacac 60 gtaatgctat 70 <210> 96 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY3673 probe <400> 96 ttatgtaaat tatcaggagt actaagccaa gcctgtcaat cgacgctgat tcctcaggat 60 gagtttgaac 70 <210> 97 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY3922 probe <400> 97 gaaatgtacc ggaatgttgg atgagaatat cgtctattcg acatttaatg ctgatcccgt 60 tgattccagc 70 <210> 98 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY0314 probe <400> 98 aagcggttgt atctgaattt aatttgtcag atataaatcg cggtggaatg acgaaagcac 60 aggcagaaaa 70 <210> 99 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY1891 probe <400> 99 aaatatctgg acctatccgc tttttgttaa taccttttca gcaaatgctc ttgtgggact 60 atcatcgtgc 70 <210> 100 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STY2356 probe <400> 100 aaataaaacg ggtgattttt ataagcccag gcaggcttat ggtgatttgg catcggtaaa 60 catggttata 70 <210> 101 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM0159 probe <400> 101 ccggtgagca cagaatattg aaaactgact ttgcattact ttgcccaaat tgtcacaaag 60 ctgttcatat 70 <210> 102 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM2743 probe <400> 102 tgacggagtt cgctttgagg acttgttttc aaaaataatg tactataagt cgccagattt 60 tcagcaggtg 70 <210> 103 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM2747 probe <400> 103 gaatccatta tgattcgccg tgtagtggta aaccagaagt aataaatgct gtgcaattat 60 tgcgctctca 70 <210> 104 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM2754 probe <400> 104 tgatttaata ggcaaaaaaa gcgaagaata tgcattaaac tgggaaaaaa accgcctaat 60 cggtctcggc 70 <210> 105 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM2761 probe <400> 105 taagcaagat tgagctgtta gagttagttc gacgtggacg tataaaattc gtcgcattcc 60 aaaatctcca 70 <210> 106 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM2762 probe <400> 106 cgtagctaat acgaaaaaag aaacagaaaa aatcggagcg acgataaagg tagtgctagg 60 cgttttcgtt 70 <210> 107 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM0654 probe <400> 107 ttattacatc ggaaaaggta tcaaacaaga ttaccagcag gcaatatact ggtttcgaaa 60 agcggcagac 70 <210> 108 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM0762 probe <400> 108 tatgcaaaaa aagaacaact tgcatgctgc catgataccg gcacttgtat cgttataatg 60 gaaattggac 70 <210> 109 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM0763 probe <400> 109 gcatttccga gagatttatc cagagatgga actctctctt gttgaagagg ggacttttgg 60 tttacatgat 70 <210> 110 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> STM0764 probe <400> 110 aaacaaccca gtcttgtttt ttcagcaacg catgctttgt ctttctcttt tgtaccgcat 60 ttgttaaaac 70 <210> 111 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> tubulin bete-4 chain probe <400> 111 aagtccagtg tctgtgatat tgcaccaaag ggtttgaaaa tggcgtctac tttcattggt 60 aactcaacct 70 <210> 112 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> actin 3 probe <400> 112 aggttcttta ccagccatct atgattggta tggagaatgc tggtatccat gaaaccacct 60 ataactccat 70 <210> 113 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Actin3-f primer <400> 113 ctcttgacta cgagcaggaa 20 <210> 114 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Actin3-r primer <400> 114 agtccttcct gatgtcgaca 20 <210> 115 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tb4-f primer <400> 115 aagaggttga cgagcagatg 20 <210> 116 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tb4-r primer <400> 116 gcctttctcc tgaacatagc 20  

Claims (7)

서열번호 31 내지 40의 올리고뉴클레오티드로 이루어진 군으로부터 선택되는 하나 이상의 올리고뉴클레오티드를 포함하는, 클로스트리디움 보툴리눔(Clostridium botulinum) 검출용 올리고뉴클레오티드 프로브;Oligonucleotide of SEQ ID NO: 31 to 40 one oligonucleotide selected from the group consisting of a nucleotide or more, including nucleotide, Clostridium botulinum (Clostridium botulinum ) oligonucleotide probe for detection; 서열번호 41 내지 50의 올리고뉴클레오티드로 이루어진 군으로부터 선택되는 하나 이상의 올리고뉴클레오티드를 포함하는, 클로스트리디움 퍼프린젠스(Clostridium perfringens) 검출용 올리고뉴클레오티드 프로브;Oligonucleotide of SEQ ID NO: 41 to 50 one oligonucleotide selected from the group consisting of a nucleotide or more, including nucleotide, Clostridium perfringens (Clostridium perfringens ) detection oligonucleotide probes; 서열번호 51 내지 60의 올리고뉴클레오티드로 이루어진 군으로부터 선택되는 하나 이상의 올리고뉴클레오티드를 포함하는, 캄필로박터 제주니(Campylobacter jejuni) 검출용 올리고뉴클레오티드 프로브; 및Campylobacter, comprising one or more oligonucleotides selected from the group consisting of oligonucleotides of SEQ ID NOs: 51-60 jejuni ) detection oligonucleotide probes; And 서열번호 61 내지 70의 올리고뉴클레오티드로 이루어진 군으로부터 선택되는 하나 이상의 올리고뉴클레오티드를 포함하는, 비브리오 파라해모리티쿠스(Vibrio parahaemolyticus) 검출용 올리고뉴클레오티드 프로브;로 이루어진 군으로부터 선택된 하나 이상의 프로브를 포함하는 식중독균 검출용 올리고뉴클레오티드 프로브 세트.Vibrio parahaemoriticus, comprising one or more oligonucleotides selected from the group consisting of oligonucleotides of SEQ ID NOs: 61-70 parahaemolyticus ) oligonucleotide probe for detection; oligonucleotide probe set for detecting food poisoning bacteria comprising at least one probe selected from the group consisting of. 제1항에 있어서, 서열번호 31 내지 70의 올리고뉴클레오티드를 포함하는, 식중독균 검출용 올리고뉴클레오티드 프로브 세트.The oligonucleotide probe set for detecting food poisoning bacteria according to claim 1, comprising oligonucleotides of SEQ ID NOs: 31 to 70. 제1항 또는 제2항에 따른 하나 이상의 올리고뉴클레오티드 프로브 세트가 고정화되어 있는 기판을 갖는 마이크로어레이.A microarray having a substrate on which at least one oligonucleotide probe set according to claim 1 is immobilized. 시료의 게놈 DNA를 제1항 또는 제2항에 따른 하나 이상의 올리고뉴클레오티드 프로브 세트와 접촉시켜 상기 시료 중의 표적 서열과 프로브 서열을 혼성화시키는 단계; 및Contacting the sample's genomic DNA with at least one oligonucleotide probe set according to claim 1 to hybridize the probe sequence with the target sequence in the sample; And 상기 프로브와 시료 중의 표적 서열 사이의 혼성화 정도를 검출하는 단계를 포함하는 식중독균을 검출하는 방법.Detecting the degree of hybridization between the probe and the target sequence in the sample. 제4항에 있어서, 상기 표적 서열은 검출 가능한 표지 물질로 표지되어 있는 것인 방법.The method of claim 4, wherein the target sequence is labeled with a detectable labeling substance. 제4항에 있어서, 상기 프로브 세트는 마이크로어레이의 기판 상에 고정화되어 있는 것인 방법.The method of claim 4, wherein the probe set is immobilized on a substrate of a microarray. 제1항 또는 제2항에 따른 하나 이상의 올리고뉴클레오티드 프로브 세트를 포함하는 식중독균 검출 키트.A food poisoning detection kit comprising at least one oligonucleotide probe set according to claim 1.
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CN102426233A (en) * 2011-11-15 2012-04-25 吉林出入境检验检疫局检验检疫技术中心 Campylobacter jejuni detection method with liquid chip
CN102507949A (en) * 2011-11-15 2012-06-20 吉林出入境检验检疫局检验检疫技术中心 Method using liquid phase chip to detect staphylococcus aureus
KR101222907B1 (en) * 2010-09-08 2013-01-16 고려대학교 산학협력단 DNA chip using random genomic DNA fragments of Yersinia enterocolitica

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KR100611056B1 (en) 2002-12-16 2006-08-10 학교법인 포항공과대학교 Oligonucleotide microchip and method for the detection of pathogenic microorganisms using same
JP2006166912A (en) 2004-11-19 2006-06-29 Hokkaido Univ Gene probe for quickly and specifically counting living food poisoning bacteria and group of biological indicator for sanitation by in situ hybridization method with cultivation

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Publication number Priority date Publication date Assignee Title
KR101222907B1 (en) * 2010-09-08 2013-01-16 고려대학교 산학협력단 DNA chip using random genomic DNA fragments of Yersinia enterocolitica
CN102426233A (en) * 2011-11-15 2012-04-25 吉林出入境检验检疫局检验检疫技术中心 Campylobacter jejuni detection method with liquid chip
CN102507949A (en) * 2011-11-15 2012-06-20 吉林出入境检验检疫局检验检疫技术中心 Method using liquid phase chip to detect staphylococcus aureus
CN102426233B (en) * 2011-11-15 2013-12-04 吉林出入境检验检疫局检验检疫技术中心 Campylobacter jejuni liquid chip detection method
CN102507949B (en) * 2011-11-15 2013-12-25 吉林出入境检验检疫局检验检疫技术中心 Method using liquid phase chip to detect staphylococcus aureus

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