KR20130046324A - The probe for cronobacter sakazakii, and the dna chip using the probe - Google Patents
The probe for cronobacter sakazakii, and the dna chip using the probe Download PDFInfo
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Abstract
Description
본 발명은 식중독 세균 Cronobacter sakazakii 탐지용 DNA 칩을 위한 올리고뉴클레오티드 탐침자 및 그를 포함한 DNA 칩에 관한 것이다.The present invention relates to an oligonucleotide probe for a DNA chip for detecting food poisoning bacteria Cronobacter sakazakii and a DNA chip comprising the same.
C. sakazakii는 신생아에게 괴사하는 전장염(necrotizing enterocolitis), 염증(sepsis), 수막염(meningitis)의 감염을 일으키는 식중독 세균으로서, 최근에는 면역 기능이 저하된 성인에게 균혈증(bacteremia), 골수염(osteomyelitis)의 감염을 일으키는 것이 보고되어 그 위험성이 강조되고 있다. C. sakazakii is a food poisoning bacterium that causes infections of necrotizing enterocolitis, inflammation, and meningitis in newborns. Recently, bacteremia and osteomyelitis in adults with reduced immune function It has been reported to cause infections, and the risk is emphasized.
식품의약품안전청의 통계자료에 따르면 매년 최대 510건의 식중독이 발생하며 약 1만여 명이 식중독 미생물에 감염된다고 한다(식약청, 식중독 통계시스템). 또한 2006년 OCAP 식품분과위원회 세미나에서 한국소비자보호원 시험검사소는 식중독으로 인한 사회 경제적 손실액이 1조 3107억원이라 밝혔다.According to the KFDA's statistics, up to 510 food poisonings occur each year and about 10,000 people are infected with food poisoning microorganisms (KFDA, food poisoning statistical system). In addition, at the 2006 OCAP Food Subcommittee Seminar, the Korea Consumer Protection Agency's test laboratory stated that the socioeconomic loss caused by food poisoning was 1.33 trillion won.
현재까지 식중독 세균의 탐지법 중에 가장 많이 사용되어온 방법은 배양법으로, 정확도는 높지만 최소 24시간 이상의 농화배양(enrichment culture)을 필요로 하며 농화배양 후에도 선택 배양, 확정 시험 단계를 필요로 하기 때문에, 이러한 전통적인 배양법은 노동 집약적이며 많은 시간이 소요된다는 단점이 있다.The most widely used method for detecting food poisoning bacteria to date is culture, which is highly accurate but requires at least 24 hours of enrichment culture, which requires selective culture and confirmed test steps even after enrichment. Traditional culture methods have the disadvantage of being labor intensive and time consuming.
이 외에도 최근에 PCR 및 항체를 이용한 면역학적 탐지법이 개발되었지만, 면역학적 방법은 실험과정이 간편한 반면 민감도가 낮고(약 105 cell/reaction), PCR 방법역시 PCR 후에 전기영동법으로 결과를 확인해야 한다는 문제점이 있다.In addition, immunological detection methods using PCR and antibodies have recently been developed. However, immunological methods are easy to perform, but have low sensitivity (about 10 5 cell / reaction). There is a problem.
또한 전통적인 배양법은 물론 PCR 및 면역학적 방법에 있어서의 가장 큰 문제점은 다수의 식중독 세균을 검출하기 위한 방법으로 적절치 않다는 것이다.In addition, the biggest problem with conventional culture methods as well as PCR and immunological methods is that it is not suitable as a method for detecting a large number of food poisoning bacteria.
한편 16S rRNA 유전자 서열을 기반으로 설계하는 탐침자(probe)의 가장 이상적인 조건은 대상 종에 속하는 여러 스트레인(strain)들을 모두 탐지하면서 타깃으로 하지 않는(non-target) 종들은 탐지하지 않는, 높은 특이성(specificity)을 갖는 것이다. 하지만 현재까지 사용되어 온 대부분의 탐침자들 중 이 조건을 충족하는 탐침자는 매우 적은 수준이다.On the other hand, the ideal condition for probes designed based on 16S rRNA gene sequences is high specificity, which detects all strains belonging to the target species but not the non-target species. It has specificity. However, among the majority of probes used to date, very few probes meet this condition.
따라서 본 발명의 목적은 식중독 세균 Cronobacter sakazakii 탐지에 있어서 특이성과 민감성이 대폭 향상된, 탐침자(probe)를 제공하는 것이다.Accordingly, an object of the present invention is to provide a probe, which has significantly improved specificity and sensitivity in detecting food poisoning bacteria Cronobacter sakazakii .
발명자들은 상기 언급한 과제를 해결하기 위해서 DNA 마이크로어레이(microarray)를 이용한 식중독 세균의 검출법을 개발하였다.The inventors have developed a method for detecting food poisoning bacteria using a DNA microarray to solve the above-mentioned problems.
발명자들은 i) 식중독 세균 C. sakazakii 의 16S rRNA 유전자를 대상으로 종-특이적 탐침자(probe) 서열을 파악하고, ii) 설계한 탐침자(probe)의 성능을 파악하기 위한 DNA 마이크로어레이 실험을 수행하여 제작된 탐침자(probe)의 특이성과 민감도를 확인하였다. 특히 기존의 DNA 마이크로어레이를 이용한 탐지법 연구와는 달리 RDP-II 데이터베이스 수록된 92만개의 16S rRNA 유전자 서열(gene sequence)들을 모두 탐침자(probe) 설계에 포함하고 대상 종에 대한 다수의 탐침자(probe)들을 사용함으로써 위양성(false positive) 및 위음성(false negative)율을 줄이는 알고리즘으로 탐지법의 정확도를 향상시켰다.The inventors conducted a DNA microarray experiment to i) identify species-specific probe sequences of the 16S rRNA gene of food poisoning bacterium C. sakazakii , and ii) determine the performance of the designed probe. The specificity and sensitivity of the prepared probes were confirmed. Unlike conventional DNA microarray detection studies, all 920,000 16S rRNA gene sequences from the RDP-II database are included in the probe design and multiple probes for the target species ( By using probes, the algorithm reduces the false positive and false negative rates and improves the accuracy of the detection method.
상기와 같은 본 발명은, 식중독 세균 Cronobacter sakazakii 탐지용 DNA 칩을 위한 올리고뉴클레오티드 탐침자로서 서열목록 40 내지 50 중 적어도 어느 하나를 포함하는 것을 특징으로 한다.The present invention as described above, characterized in that it comprises at least one of SEQ ID NO: 40 to 50 as an oligonucleotide probe for the DNA chip for detecting food poisoning bacteria Cronobacter sakazakii .
본 발명은 또한 서열목록 40 내지 50 중 적어도 어느 하나의 탐침자를 포함하는 식중독 세균 Cronobacter sakazakii 탐지용 DNA 칩인 것을 특징으로 한다.The present invention is also characterized in that the DNA chip for detecting food poisoning bacteria Cronobacter sakazakii comprising at least one probe of SEQ ID NO: 40 to 50.
본 발명에 따르면, 종래 방법에 비하여 식중독 세균 Cronobacter sakazakii 를 훨씬 신속하고도 정확하게 탐지하는 게 가능해진다.According to the present invention, it becomes possible to detect food poisoning bacteria Cronobacter sakazakii much more quickly and accurately than the conventional method.
도면 1. 타깃 분류군(Target taxon)의 계통수(phylogenetic tree)
도면 2. 후보 올리고뉴클레오티드 서열(각각 서열목록 1 내지 50으로 첨부함)
도면 3. (상) 후보 oligonucleotide 탐침자(probe) 중 cutoff 40 이상의 S/N ratio
(하) 후보 oligonucleotide 탐침자(probe)의 S/N ratio
도면 4. 특이성이 확인된 올리고뉴클레오티드 서열(서열목록 40 내지 50)Figure 1. Phylogenetic tree of the target taxon
Candidate oligonucleotide sequences (attached as SEQ ID NOS: 1-50, respectively)
Figure 3. S / N ratio of
S / N ratio of candidate oligonucleotide probe
Figure 4. Oligonucleotide sequence confirmed specificity (SEQ ID NO: 40 to 50)
이하 본 발명을 구체적인 실시예와 함께 상세히 설명한다.Hereinafter, the present invention will be described in detail with specific examples.
＜재료 및 방법＞<Materials and methods>
1. 균주1. Strain
본 발명에서 사용된 Cronobacter sakazakii strain은 ATCC(American Type Culture collection)으로부터 얻은 것이다(ATCC29544). Cronobacter 균주는 DifcoTM Nutrient Agar에서 계대배양 하였으며 conventional PCR의 주형(template)으로 사용할 게노믹 DNA(genomic DNA)의 추출은 Sambrook과 Russell의 방법을 따랐다. Cronobacter sakazakii strain used in the present invention is obtained from the American Type Culture collection (ATCC) (ATCC29544). Cronobacter strains were subcultured in Difco TM Nutrient Agar, and extraction of genomic DNA for use as a template for conventional PCR was followed by Sambrook and Russell.
2. 2. C. sakazakiiC. sakazakii 의 계통분석학적 위치 파악 및 탐침자(probe) 선별Phylogenetic localization and probe selection
16S rRNA 유전자 서열을 이용한 식중독 세균의 검출용 DNA 칩 제작에 필요한 탐침자(probe)를 디자인하기 위해, RDP II 데이터베이스(버전 10)에서 759,000개의 16S rRNA 유전자 서열(gene sequence)을 검색했다. 759,000개의 16S rRNA 유전자 서열(gene sequence) 중에서 분리(isolate) 되었으며 사이즈가 1.2 kb 이상인 기준에 적합한 서열(sequence)을 선택한 후 pintail algorithm으로 sequence quality를 확인하였다.In order to design probes for the production of DNA chips for the detection of food poisoning bacteria using 16S rRNA gene sequences, 759,000 16S rRNA gene sequences were searched in the RDP II database (version 10). The sequence quality was isolated among 759,000 16S rRNA gene sequences, and the sequence quality was determined by pintail algorithm.
선택된 서열을 정렬(alignment)함으로써 각 탐침자(probe) 그룹별로 공통 배열(consensus sequence)을 확보하며, 서열 보존부위 중에서 모호하지 않은(unambiguous) 부분을 탐침자(probe) 디자인에 사용했다.By aligning the selected sequences, a consensus sequence was obtained for each probe group, and an unambiguous portion of the sequence conserved region was used for the probe design.
계통수(phylogenetic tree)를 작성함으로써 선정된 서열(sequence)의 진화적 거리(evolutionary distance)를 확인하였으며 탐침자(probe) 그룹 내에서, 탐침자(probe) 그룹 사이의, 관련된 종(species) 간에, 관련된 속(genera)에 대해 진화적 거리(evolutionary distance)를 계산했다.By constructing a phylogenetic tree, the evolutionary distances of the selected sequences were identified and, within the probe group, between the probe groups, between related species, Evolutionary distances were calculated for the genera involved.
3. PCR-라벨링(Labeling)3. PCR-Labeling
칩 혼성화(hybridization)에 사용될 PCR 주형(template)은 PCR 중에 Cy3-dUTP로 라벨링 하였다. 1μl의 주형 DNA, Cy3-dUTP 0.5 μl, LA Taq 1μl, 25μl의 2X GC buffer I, dNTPs 8μl, 0.5μl(5pmole)씩의 PCR primer(8F, 1392R, 23F, 1492R), 1μl의 BSA(20mg/ml, Bovine Serum Albumin, Roche, Germany)에 12μl의 증류수를 넣은 총 50μl의 PCR mixture로 PCR을 수행하였다. PCR 반응은 95℃에서 5분간 초기 변성기 후에, 95℃에서 30초간 변성-50℃에서 30초간 결합(annealing)-72℃에서 1분 30초간 신장(extension)하는 과정을 35회 반복하였다. PCR thermocycler는 GeneAmp PCR System 9700(Applied Biosystems, Calif. USA)을 사용하였다.PCR templates to be used for chip hybridization were labeled with Cy3-dUTP during PCR. 1 μl template DNA, Cy3-dUTP 0.5 μl,
4. 칩 혼성화(chip hybridization)4. Chip hybridization
DNA 혼성화(hybridization)에는 CombiMatrix사의 CustomArray 4X2 칩을 사용하였다. 30μl의 nuclease-free water로 65℃의 hybridization chamber에서 10분간 인큐베이션(incubation)한 후 30μl의 pre-hybridization solution으로 50℃의 hybridization chamber에서 30분간 인큐베이션(incubation)하였다. Pre-hybridization solution을 제거한 뒤 정제된(QIAquick PCR purification kit) PCR product를 포함한 hybridization solution을 넣고 16시간 동안 혼성화 하였다.CombiMatrix's CustomArray 4X2 chip was used for DNA hybridization. After incubation for 10 minutes in a hybridization chamber at 65 ℃ with 30μl nuclease-free water (incubation) for 30 minutes in a hybridization chamber at 50 ℃ with 30μl pre-hybridization solution. After removing the pre-hybridization solution, the hybridization solution containing the purified (QIAquick PCR purification kit) PCR product was added and hybridized for 16 hours.
＜결과＞<Result>
1. One. C. sakazakiiC. sakazakii 의 계통분석학적 위치 파악 및 탐침자(probe) 선별Phylogenetic localization and probe selection
50개의 서열을 정렬(alignment)하여 계통수(phylogenetic tree)를 그려봄으로써 선정된 서열의 진화적 거리(evolutionary distance)를 확인하였다(도면 1). DE (evolutionary distance)가 99.5％ 이하인 것을 기준으로 OTU(operational taxonomic unit)를 결정하였다. 진화적 거리(evolutionary distance)는 탐침자(probe) 그룹 내에서, 탐침자(probe) 그룹 사이의, 관련된 종(species) 간에, 관련된 속(genera)에 대해 각각 계산되었다.The evolutionary distance of the selected sequence was confirmed by drawing a phylogenetic tree by aligning 50 sequences (Fig. 1). An operational taxonomic unit (OTU) was determined based on an evolutionary distance (DE) of 99.5% or less. Evolutionary distances were calculated for the related genera, within the probe group, between the probe groups, between related species, and respectively.
그 결과 탐침자(probe) 그룹 내의 진화적 거리(evolutionary distance)는 0.0027이었고 편차는 0.0008, 관련된 종(species)과의 진화적 거리(evolutionary distance)는 0.0166 편차는 0.0025이었고 관련된 속(genera)과의 진화적 거리(evolutionary distance)는 0.0403 편차는 0.0039으로 계산되었다.As a result, the evolutionary distance within the probe group was 0.0027, the deviation was 0.0008, the evolutionary distance from the related species was 0.0166, the deviation was 0.0025, and the associated genera The evolutionary distance was calculated to be 0.039 with a 0.0403 deviation.
수집된 서열을 정렬(alignment)함으로써 공통 배열(consensus sequence)을 찾을 수 있었고 일정하게 보존되어 있는 공통 배열(consensus sequence) 중에서도 오직 모호하지 않은(unambiguous) 지역을 탐침자(probe) 디자인에 사용하였다.Consensus sequences could be found by aligning the collected sequences, and only unambiguous regions were used in probe design, among conserved consensus sequences.
설계 가능한 25-mer oligonucleotide는 1149개로 확인되었으며 Tm(melting temperature)은 66.2 ± 3.2, GC content[(G+C)％]는 55.8 ± 7.9 이었다. 이 중 32개의 탐침자(probe)가 Primrose software를 이용한 생물정보학적 분석을 통해 타깃 종(target species, C. sakazakii)에 특이성이 있는 것으로 확인되었으며 타깃이 아닌(non-target) Dichelobacter nodo년, Nevskia ramosa, Citrobacter rodentium을 동시에 탐지하는 탐침자(probe)는 8개로 확인되었다. 나머지 탐침자(probe)들은 non-target을 4개 이상 탐지하는 것으로 확인되었다.The designable 25-mer oligonucleotides were identified as 1149, and the melting temperature (Tm) was 66.2 ± 3.2 and the GC content [(G + C)%] was 55.8 ± 7.9. Thirty-two probes were identified for specific target species ( C. sakazakii ) by bioinformatics analysis using Primrose software, and non-target Dichelobacter nodo, Nevskia. Eight probes were identified that simultaneously detected ramosa and Citrobacter rodentium . The remaining probes were found to detect more than four non-targets.
2. DNA 칩 제작 및 특이성(specificity) 검증2. DNA chip fabrication and specificity verification
발굴된 모든 탐침자(probe) 서열들을 이용하여 Combimatrix사의 CombiChip 또는 올리고뉴클레오티드(oligonucleotide) DNA 칩을 제작하였다. 제작된 DNA 칩은 타깃 미생물에 특이적인 PCR로부터 얻어진 PCR 산물(product)과 혼성화(hybridization)하여 타깃 미생물의 검출/동정에 필요한 특이성을 갖추었는지를 검증한다.CombiChip's CombiChip or oligonucleotide DNA chips were prepared using all probe sequences. The produced DNA chip hybridizes with a PCR product obtained from PCR specific to the target microorganism and verifies whether the target DNA has the specificity necessary for detection / identification of the target microorganism.
타깃 DNA는 16S rRNA 유전자에 대한 universal primer를 이용하여 PCR 증폭하며, PCR 증폭 반응 중에 형광감쇄성이 낮은 Cy3를 이용하여 표지하며, 혼성화 반응 후, 형광신호를 수집하여 음성대조군과 반응하지 않으며 배경 신호에 비해 통계학적으로 유의미한 수준 이상의 형광신호를 발생하는지 여부에 따라 제작된 DNA 칩의 특이성을 C. sakazakii에 대하여 평가하였다.Target DNA is PCR amplified using universal primer for 16S rRNA gene, labeled using Cy3 with low fluorescence attenuation during PCR amplification reaction, and after hybridization reaction, it collects fluorescent signal and does not react with negative control and does not react with background signal C. sakazakii was evaluated for the specificity of the DNA chip fabricated according to whether or not to generate a fluorescence signal more than statistically significant level.
3. 선별된 탐침자(probe)의 성능 확인3. Check the performance of selected probes
제작된 50개의 탐침자(도면 2)의 성능은 DNA 혼성화(hybridization)에서 발생한 형광 신호값을 노이즈 시그널(noise signal)의 평균값으로 나누어 준 S/N 값으로 확인하였다. 50개의 탐침자(probe) 서열은 칩 상에 triplicate로 spotting 되었으며, 발생한 시그널(signal)은 그 평균값을 사용하였다.The performance of the 50 probes (Fig. 2) produced was confirmed by the S / N value obtained by dividing the fluorescence signal value generated by DNA hybridization by the average value of the noise signal. Fifty probe sequences were spotted on the chip as triplicate, and the average signal generated was used.
50개 탐침자(probe)의 S/N 값 중 유의미한 수준의 값을 기준(cutoff)으로 하여 탐침자(probe)의 성능을 평가하였으며, 그 결과 11개의 탐침자(probe)가 C. sakazakii의 탐지에 특이적인 탐침자(probe)임을 확인하였다(도면 3 및 도면 4).The performance of the probe was evaluated based on the significant level of the S / N values of the 50 probes. As a result, 11 probes detected C. sakazakii . It was confirmed that the probe is specific to (Fig. 3 and 4).
이로써 종래 방법에 비하여 식중독 세균 Cronobacter sakazakii를 훨씬 신속하고도 정확하게 탐지할 수 있게 된다.This makes it possible to detect food poisoning bacteria Cronobacter sakazakii much more quickly and accurately than the conventional method.
Claims (2)
식중독 세균 Cronobacter sakazakii 탐지용 DNA 칩을 위한 올리고뉴클레오티드 탐침자.Characterized in that at least one of SEQ ID NO: 40 to 50,
Oligonucleotide probe for DNA chip for detecting food poisoning bacteria Cronobacter sakazakii .
식중독 세균 Cronobacter sakazakii 탐지용 DNA 칩.Characterized in that it comprises at least one probe of SEQ ID NO: 40 to 50,
DNA chip for detecting food poisoning bacteria Cronobacter sakazakii .
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