KR101332366B1 - Primer set and method for distinguishing of Bacillus thuringiensis and Bacillus cereus - Google Patents
Primer set and method for distinguishing of Bacillus thuringiensis and Bacillus cereus Download PDFInfo
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Abstract
본 발명은 바실러스 튜린겐시스(Bacillus thuringiensis), 바실러스 세레우스(Bacillus cereus) 구별용 프라이머 셋트 및 이를 이용한 바실러스 튜린겐시스, 바실러스 세레우스 식별 방법에 관한 것이다.
또한, 본 발명은 상기 프라이머 셋트를 포함한 바실러스 튜린겐시스, 바실러스 세레우스 의 구별용 키트 및 조성물에 관한 것으로 본 발명에 의하면 식중독균인 바실러스 세레우스 와 미생물 농약균주인 바실러스 튜린겐시스를 신속하고 정확하게 구별해 낼 수 있다. The present invention Bacillus thuringiensis ( Bacillus thuringiensis), Bacillus cereus (Bacillus cereus ) and a primer set for distinguishing and Bacillus thuringiensis, Bacillus cereus identification method using the same.
In addition, the present invention relates to a kit and a composition for distinguishing Bacillus thuringiensis, Bacillus cereus comprising the primer set according to the present invention Bacillus cereus and The bacterium strain Bacillus thuringiensis can be identified quickly and accurately.
Description
본 발명은 미생물 농약 균주인 바실러스 튜린겐시스 및 식중독균 바실러스 세레우스를 신속하고 정확하게 구별할 수 있는 프라이머 셋트 및 이를 포함하는 조성물, 키트에 관한 것이다. 보다 상세하게는 서열번호 2 및 3, 서열번호 5 및 6의 프라이머 셋트에 관한 것으로 이를 이용하여 바실러스 튜린겐시스와 바실러스 세레우스를 동시에 신속하게 구별할 수 있는 프라이머 셋트 및 구별 방법에 관한 것이다.
The present invention relates to a set of primers and compositions, kits comprising the same, which can quickly and accurately distinguish between bacterium Bacillus thuringiensis and food poisoning bacillus Bacillus cereus. More specifically, the present invention relates to primer sets of SEQ ID NOS: 2 and 3, SEQ ID NOs: 5 and 6, and to primer sets and methods for quickly distinguishing Bacillus thuringiensis and Bacillus cereus simultaneously.
바실러스 세레우스는 포자형성 세균으로 자연계에 흔히 존재하고 식중독을 일으키며 포자는 저항성이 강하여 일반적인 식품제조 및 조리과정에서는 사멸하지 않는다. 따라서 식품의약품안전청에서는 식품의 종류에 따라 바실러스 세레우스의 허용규격을 설정하여 관리하고 있다. 바실러스 튜린겐시스 역시 포자형성세균이고 특이하게도 해충에 선택적인 살충성 독소를 생성하므로 친환경적인 미생물농약으로 근래에 그 사용량이 점차 증가하고 있다. 따라서 식품의 원부재료로 사용되는 농작물에 바실러스 튜린겐시스가 잔류할 가능성은 충분히 높다. Bacillus cereus is a spore-forming bacterium that is common in nature and causes food poisoning. Spores are resistant and do not die in general food manufacturing and cooking processes. Therefore, the Food and Drug Administration sets and manages the acceptable standards of Bacillus cereus according to the type of food. Bacillus thuringiensis is also a spore-forming bacterium and specifically produces pesticidal toxins that are selective for pests. Therefore, there is a high possibility that Bacillus thuringiensis remains in crops used as raw materials of food.
식품에 친환경적 미생물 농약으로 사용된 바실러스 튜린겐시스의 잔존이 식중독균으로 오인될 수 있는 문제점이 상존하므로 구별 검출의 중요성이 필요함에도 불구하고 바실러스 세레우스와 바실러스 튜린겐시스 는 생화학적으로나 유전적으로 매우 유사하여 식품공전상의 생화학적 방법으로는 두 균주를 구분하기 어려운 실정이다. Although there is a problem that Bacillus thuringiensis used as an eco-friendly microbial pesticide in food may be mistaken for food poisoning bacteria, Bacillus cereus and Bacillus thuringiensis are very similar in biochemistry and genetics despite the importance of distinctive detection. Therefore, it is difficult to distinguish between two strains by biochemical methods in food industry.
현재 바실러스 튜린겐시스를 확인하기 위해서는 포자를 형성시켜 살충독소를 관찰하는 현미경 검사법을 시행하여야 한다. 하지만 이 방법은 많은 시간, 노력, 비용이 소요되는 단점이 있으며, 특히 crystal protein을 구분하는데 있어서 많은 혼동을 일으킨다는 문제점을 가지고 있다. Currently, to determine Bacillus thuringiensis, it is necessary to perform microscopic examination of spore formation to observe pesticidal toxin. However, this method is disadvantageous in that it takes a lot of time, effort, and cost, and in particular, causes a lot of confusion in identifying crystal proteins.
근래 유기농산물을 포함한 친환경농산물을 찾는 소비자가 늘어남에 따라 미생물농약의 사용증가로 식품에 잔류하는 바실러스 튜린겐시스가 바실러스 세레우스로 오인되어 식품규격을 초과하는 사례가 발생하고 있으며 이는 경제적 손실로 이어질 수 있어 보다 간편하고 신속한 검출법의 개발이 필요한 실정이다. Recently, as more consumers search for eco-friendly agricultural products, including organic produce, Bacillus thuringiensis, which remains in food due to increased use of microbial pesticides, is mistaken for Bacillus cereus, resulting in exceeding food standards. It is necessary to develop a simpler and faster detection method.
특히, 현재 시판중인 식중독 검출키트 중 바실러스 세레우스에 특이적인 프라이머 셋트는 바실러스 튜린겐시스와 바실러스 세레우스를 구분없이 검출한다는 문제점을 가지고 있다. In particular, a primer set specific to Bacillus cereus among currently available food poisoning detection kits has a problem of detecting Bacillus thuringiensis and Bacillus cereus without distinction.
이에 따라 미생물 농약 균주인 바실러스 튜린겐시스와 식중독균인 바실러스 세레우스를 신속하고 정확하게 구별할 수 있는 방법이 절실히 필요한 상황이다. Accordingly, there is an urgent need for a method for quickly and accurately distinguishing Bacillus thuringiensis, a microbial pesticide strain, and Bacillus cereus, a food poisoning bacterium.
국내에서 미생물농약균주인 바실러스 튜린겐시스와 식중독균인 바실러스 세레우스를 동시에 구분하는 multiplex PCR 및 multiplex real-time PCR법은 아직 보고된 바 없다.In Korea, no multiplex PCR and multiplex real-time PCR methods for simultaneously distinguishing bacterium Bacillus thuringiensis and food poisoning bacillus Bacillus cereus have been reported.
이에 본 발명자들은 매우 유사한 두 종에 대하여 특이적인 프라이머 셋트와 TaqMan probe를 제작하여 multiplex PCR 및 multiplex real-time PCR 방법을 개발함으로써 미생물농약균주인 바실러스 튜린겐시스를 식중독균인 바실러스 세레우스와 신속하게 구분 검출 할 수 있는 시스템을 발명하기에 이르렀다.
In this regard, the present inventors developed a multiplex PCR and multiplex real-time PCR method by constructing a specific primer set and TaqMan probe for two very similar species, thereby rapidly distinguishing bacterium strain Bacillus thuringiensis from food poisoning bacterium Bacillus cereus. Invented a system that can detect.
본 발명의 목적은 미생물 농약 균주인 바실러스 튜린겐시스 및 식중독균 바실러스 세레우스를 신속하고 정확하게 구별할 수 있는 프라이머 셋트 및 이를 포함하는 조성물, 키트를 제공하는데 있다. It is an object of the present invention to provide a set of primers and compositions, kits comprising the same, which can quickly and accurately distinguish between Bacillus thuringiensis and food poisoning bacillus Bacillus cereus, which are microbial pesticide strains.
또한 본 발명의 목적은 상기 프라이머 셋트를 이용하여 바실러스 튜린겐시스와 바실러스 세레우스를 식별할 수 있는 방법을 제공하는 것이며, 이를 통해 신속하고 정확하게 두 균주를 구별 가능토록 함을 목적으로 한다.
It is also an object of the present invention to provide a method for distinguishing Bacillus thuringiensis and Bacillus cereus using the primer set, and to make it possible to distinguish between two strains quickly and accurately.
상기와 같은 목적을 달성하기 위해서, 본 발명은 바실러스 튜린겐시스(Bacillus thuringiensis)와 바실러스 세레우스(Bacillus cereus)의 groEL(서열번호1)의 증폭을 위한 프라이머 셋트 및 바실러스 튜린겐시스의 cry유전자(서열번호4)의 증폭을 위한 프라이머 셋트를 포함하는 바실러스 튜린겐시스, 바실러스 세레우스 구별용 프라이머 셋트를 제공한다. In order to achieve the above object, the present invention is Bacillus tube ringen sheath (Bacillus thuringiensis), and Bacillus cereus groEL (SEQ ID NO: 1) primer set and Bacillus tube cry gene of ringen System for the amplification of the (Bacillus cereus) ( A primer set for distinguishing Bacillus thuringiensis and Bacillus cereus, comprising a primer set for amplification of SEQ ID NO: 4) is provided.
또한 본 발명은 상기와 같은 프라이머 셋트를 포함하는 바실러스 튜린겐시스, 바실러스 세레우스 구별용 조성물을 제공한다.In another aspect, the present invention provides a composition for distinguishing Bacillus thuringiensis, Bacillus cereus comprising a primer set as described above.
또한 본 발명은 상기와 같은 프라이머 셋트를 포함하는 바실러스 튜린겐시스, 바실러스 세레우스 구별용 키트를 제공한다.In another aspect, the present invention provides a kit for distinguishing Bacillus thuringiensis, Bacillus cereus comprising the primer set as described above.
또한 본 발명은 상기의 프라이머 셋트를 이용한 바실러스 튜린겐시스와 바실러스 세레우스 식별 방법을 제공한다.
The present invention also provides a method for identifying Bacillus thuringiensis and Bacillus cereus using the primer set described above.
본 발명에 따르면, 생화학적, 유전적으로 매우 유사하여 종래 생화학적인 방법으로 신속하고 정확하게 검출이 어려웠던 친환경 미생물 농약 균주인 바실러스 투린겐시스와 식중독균인 바실러스 세레우스를 검출능을 향상시키면서 빠르게 구별해 낼 수 있다.
According to the present invention, it is possible to quickly distinguish between Bacillus thuringiensis, an environmentally friendly microbial pesticide strain, and Bacillus cereus, a food poisoning bacterium, which are very biochemically and genetically similar and difficult to detect quickly and accurately by conventional biochemical methods. have.
도1은 식중독균인 바실러스 세레우스와의 구분검출을 위한 미생물농약균주 바실러스 튜린겐시스에 특이적인 정방향 프라이머 서열의 blast결과를 도시한다.
도2는 식중독균인 바실러스 세레우스와의 구분검출을 위한 미생물농약균주 바실러스 튜린겐시스에 특이적인 역방향 프라이머 서열의 blast결과를 도시한다.
도면 3은 본 발명에서 제작한 프라이머 셋트를 이용하여 multiplex conventional PCR 반응을 수행하여 미생물 농약 분리주 8주와 Positive Control인 바실러스 튜린겐시스 1주, 바실러스 세레우스 1주, Negative Control을 포함한 총 11주를 대상으로 직접 검출한 결과를 도시한다.
도면 4는 본 발명에서 제작한 프라이머 셋트와 TaqMan probe를 이용하여 multiplex real-time PCR 반응을 수행하여 미생물 농약 분리주 8주와 Positive Control인 바실러스 튜린겐시스 1주, 바실러스 세레우스 1주, Negative Control을 포함한 총 11주를 대상으로 직접 검출한 결과를 도시한다.
도면 5는 Non-target Strain인 Enterobactor sakazakii , E. coli 0157: H7 , Listeria grayi , Salmonella enterilidis , Shigella dysenteria , Staphylococcus aureus , Vibrio vulnificus , Yersinia ruckeri, Bacillus subtilis , Bacillus amyloloquefaciens의 DNA와 Positive Control인 바실러스 튜린겐시스 1주, 바실러스 세레우스 1주, Negative Control을 포함한 총 13주를 대상으로 본 발명에서 제작한 Primer set와 Taqman probe의 특이도를 검사한 결과를 도시한다.
도면 6은 미생물농약균주 바실러스 튜린겐시스인 ATCC13366 균주의 생균수 측정 후, 십진 희석 하여 real-time PCR을 통해 검출한계를 검사한 결과이다. (2.2×105 cfu/rxn~2.2×101 cfu/rxn)
도면 7은 식중독균 바실러스 세레우스인 ATCC 21772 균주의 생균수 측정 후, 십진 희석하여 real-time PCR을 통해 검출한계를 검사한 결과이다. (3.2×105 cfu/rxn ~ 3.2×101 cfu/rxn)Figure 1 shows the blast results of the forward primer sequence specific for the microbial pesticide strain Bacillus thuringiensis for detection of the food poisoning bacteria Bacillus cereus.
Figure 2 shows the blast results of the reverse primer sequence specific for the microbial pesticide strain Bacillus thuringiensis for the detection and detection of food poisoning bacteria Bacillus cereus.
3 shows a total of 11 weeks including 8 strains of microbial pesticide isolates, 1 strain of Bacillus thuringiensis, 1 strain of Bacillus cereus, and 1 Negative Control by performing multiplex conventional PCR reactions using the primer set prepared in the present invention. The results detected directly by the object are shown.
4 is a microbial pesticide isolate 8 weeks and positive control Bacillus thuringiensis 1 week, Bacillus cereus 1 week, Negative Control by performing multiplex real-time PCR reaction using the primer set and TaqMan probe prepared in the present invention The results of direct detection for a total of 11 weeks, including, are shown.
5 is a non-target strainEnterobactor sakazakii , E. coli 0157: H7 , Listeria grayi , Salmonella enterilidis , Shigella dysenteria , Staphylococcus aureus , Vibrio vulnificus , Yersinia ruckeri, Bacillus subtilis , Bacillus amyloloquefaciensThe results of testing the specificity of the Primer set and Taqman probe prepared in the present invention for 13 weeks including DNA and positive control Bacillus thuringiensis 1 week, Bacillus cereus 1 week, Negative Control.
6 is a result of measuring the limit of detection by real-time PCR after diluting the number of viable cells of the ATCC13366 strain of microbial pesticide strain Bacillus thuringiensis. (2.2 × 105 cfu / rxn ~ 2.2 × 10One cfu / rxn)
7 is a result of measuring the detection limit through real-time PCR after diluting the number of viable cells of ATCC 21772 strain of food poisoning bacterium Bacillus cereus. (3.2 × 105 cfu / rxn to 3.2 × 10One cfu / rxn)
본 발명을 실시하기 위해서 구별 대상인 바실러스 세레우스와 바실러스 튜린겐시스의 유전자를 분석하여 두 균주에 공통으로 포함되어 있는 유전자와 두 균주 중 하나의 균주에만 특이적으로 있는 유전자를 선별한다. 보다 상세하게는 두 균주에 공통으로 존재하는 유전자는 groEL(서열번호1) 유전자 일 수 있으며, 하나의 균주에만 특이적으로 존재하는 유전자는 cry유전자(서열번호4)일 수 있다. In order to practice the present invention, genes of Bacillus cereus and Bacillus thuringiensis, which are distinguished from each other, are analyzed to select genes commonly included in both strains and genes specific to only one of the two strains. More specifically, the gene commonly present in both strains may be a groEL (SEQ ID NO: 1) gene, and a gene specifically present in only one strain may be a cry gene (SEQ ID NO: 4).
본 발명에 사용된 용어인 “프라이머”란 짧은 자유 3말단 수산화기(free 3'-hydroxyl group)를 가지는 핵산 서열로 상보적인 주형(template)과 염기쌍(base pair)을 형성할 수 있고 주형 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응을 위한 시약(DNA 중합효소 또는 역전사 효소) 및 상이한 4가지 dNTP(deoxynucleoside triphospate)의 존재 하에서 DNA합성을 개시할 수 있다.The term "primer" used in the present invention is a nucleic acid sequence having a short free 3'-hydroxyl group, which can form complementary templates and base pairs, and By a short nucleic acid sequence that serves as a starting point for. Primers can initiate DNA synthesis in the presence of reagents for polymerisation (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphospates (dNTPs) at appropriate buffers and temperatures.
본 발명에서 프라이머 쌍은 표적 유전자 서열을 인지하는 정방향 및 역방향의 프라이머로 이루어진 모든 조합의 프라이머 쌍을 포함하나, 바람직하게는, 특이성 및 민감성을 가지는 분석 결과를 제공하는 프라이머 쌍이다.Primer pairs in the present invention include primer pairs of all combinations of forward and reverse primers that recognize the target gene sequence, but are preferably primer pairs that provide assay results with specificity and sensitivity.
따라서, 본 발명의 프라이머 셋트는 groEL(서열번호1) 유전자 및 cry유전자(서열번호4)에 대해 특이적인 프라이머로 제한 없이 구성될 수 있다. 바람직하게는 각각 서열번호 2 및 3을 갖는 프라이머 셋트와 서열 번호 5 및 6을 갖는 프라이머 셋트로 구성될 수 있다. Thus, the primer set of the present invention can be configured without restriction to primers specific for the groEL (SEQ ID NO: 1) gene and cry gene (SEQ ID NO: 4). Preferably, it may consist of a primer set having SEQ ID NOs: 2 and 3 and a primer set having SEQ ID NOs: 5 and 6, respectively.
또한 본 발명의 프라이머 핵산 서열은 필요한 경우, 분광학적, 광화학적, 생화학적, 면역화학적 또는 화학적 수단에 의해 직접적으로 또는 간접적으로 검출 가능한 표지를 포함할 수 있다. 표지의 예로는, 효소(예를 들어, 호스래디쉬 퍼옥시다제, 알칼린 포스파타아제), 방사성 동위원소, 형광성 분자, 화학그룹(예를 들어, 바이오틴) 등이 있다.
In addition, the primer nucleic acid sequence of the present invention may include a label which can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means, if necessary. Examples of labels include enzymes (eg horseradish peroxidase, alkaline phosphatase), radioisotopes, fluorescent molecules, chemical groups (eg biotin), and the like.
본 발명은 groEL(서열번호1) 유전자 및 cry유전자(서열번호4)에 대해 특이적인 프라이머 셋트를 포함하는 바실러스 튜린겐시스와 바실러스 세레우스 구별용 조성물 및 키트를 제공한다. The present invention provides compositions and kits for distinguishing Bacillus thuringiensis and Bacillus cereus, comprising primer sets specific for the groEL (SEQ ID NO: 1) gene and the cry gene (SEQ ID NO: 4).
본 발명의 진단용 키트 및 진단용 조성물은, 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성 성분 조성물, 용액 또는 장치를 더 포함할 수 있다. 바람직하게는, 상기 구별용 키트 및 조성물은 역전사 반응 및 실시간 다중 PCR(real-time multiplex PCR)을 수행하기 위해 필요한 필수 요소를 포함하는 바실러스 튜린겐시스와 바실러스 세레우스 구별용 조성물 및 키트이다. 바람직하게는 본 발명의 조성물 및 키트는 서열번호 2 및 3의 프라이머 셋트, 서열번호 5 및 6의 프라이머 셋트를 포함할 수 있다.The diagnostic kit and diagnostic composition of the present invention may further comprise one or more other component compositions, solutions or devices suitable for the assay method. Preferably, the distinguishing kit and composition is a composition and kit for distinguishing Bacillus thuringiensis and Bacillus cereus comprising essential elements necessary for performing reverse transcription reaction and real-time multiplex PCR. Preferably the compositions and kits of the invention may comprise a primer set of SEQ ID NOs: 2 and 3, a primer set of SEQ ID NOs: 5 and 6.
본 발명의 진단용 키트 및 진단용 조성물은, 검출 유전자에 대한 특이적인 각각의 프라이머 쌍 외에도 상보적인 DNA를 증폭하기 위한 DNA 중합효소, 튜브 또는 다른 적절한 컨테이너, 반응 완충액(pH 및 마그네슘 농도는 다양), 데옥시뉴클레오타이드(dNTPs), Hot start Taq-폴리머라아제 및 역전사 효소와 같은 효소, DNase, RNase 억제제, DEPC-수(DEPC-water), 멸균수 등을 포함할 수 있다.
The diagnostic kits and diagnostic compositions of the present invention comprise DNA polymerases, tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), for amplifying complementary DNA, in addition to each primer pair specific for the detection gene. Enzymes such as oxynucleotides (dNTPs), Hot start Taq-polymerases and reverse transcriptases, DNases, RNase inhibitors, DEPC-water, sterile water and the like.
또한, 바람직하게는 본 발명의 구별용 키트 및 구별용 조성물은 특이적 결합을 이룰 수 있는 프로브를 포함할 수 있다. In addition, preferably, the discriminating kit and the distinguishing composition of the present invention may include a probe capable of achieving a specific binding.
본 발명에서 “프로브”란, 특이적 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링 되어 있어서 특정 mRNA의 존재 유무를 확인할 수 있다. 프로브는 올리고뉴클레오타이드(oligonucleotide) 프로브, 단쇄 DNA(single stranded DNA) 프로브, 이중쇄 DNA(double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 또한, 실시간 PCR을 수행하기 위해서는 형광표식 프로브를 이용할 수 있다. 보다 구체적으로 TaqMan 프로브를 이용하는 경우 5' 말단은 형광물질로 3' 말단은 quencher 물질로 수식한 올리고뉴클레오타이드를 PCR 반응액에 첨가할 수 있다. 또한, Cycling 프로브를 이용한 실시간 PCR은 RNA와 DNA로 이루어지는 키메라 프로브와 RNAse H로 구성된 고감도의 검출방법이다. 이 역시 프로브의 5' 말단은 형광물질로 3' 말단은 quencher 물질로 표식할 수 있다.In the present invention, the term "probe" refers to nucleic acid fragments such as RNA or DNA corresponding to short bases to hundreds of bases that can achieve specific binding, and are labeled to confirm the presence or absence of specific mRNAs. Probes may be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like. In addition, in order to perform real-time PCR, a fluorescent labeling probe may be used. More specifically, when the TaqMan probe is used, oligonucleotides modified at 5 'end with fluorescent material and 3' end with quencher material may be added to the PCR reaction solution. In addition, real-time PCR using Cycling probe is a high sensitivity detection method consisting of a chimeric probe consisting of RNA and DNA and RNAse H. Again, the 5 'end of the probe can be labeled with a fluorescent material and the 3' end with a quencher material.
본 발명의 일 실시예에서는 바람직하게는 cry유전자를 타깃으로 하는 프로브(서열번호7), groEL유전자를 타깃으로하는 프로브(서열번호8)을 제시하고 있으나 이에 제한되는 것은 아니며 상기 프로브를 형광 물질로 표식할 수 있다. 이는 당업계에 공지된 형광물질을 제한없이 사용할 수 있다. 본 발명의 일 실시예에서 바람직하게는 5’말단을 FAM 또는 HEX로 표시하고, BHQ1, BHQ2등으로 표시할 수 있음을 제시하고 있으나 이러한 실시예에 제한되는 것은 아니다. In one embodiment of the present invention, but preferably a probe targeting the cry gene (SEQ ID NO: 7), a probe targeting the groEL gene (SEQ ID NO: 8), but is not limited thereto. It can be marked. It can be used without limitation fluorescent materials known in the art. In an embodiment of the present invention, it is preferable that the 5 'end is displayed as FAM or HEX, and it can be displayed as BHQ1, BHQ2, etc., but is not limited thereto.
또한 본 발명의 일 양태로써, groEL(서열번호1) 유전자 및 cry유전자(서열번호4)에 대해 특이적인 프라이머 셋트를 이용한 바실러스 세레우스와 바실러스 튜린겐시스 식별 방법을 제공한다. In another aspect, the present invention provides a method for identifying Bacillus cereus and Bacillus thuringiensis using a primer set specific for the groEL (SEQ ID NO: 1) gene and the cry gene (SEQ ID NO: 4).
본 발명의 방법에 있어서, 상기 방법에 의한 증폭 산물의 검출은 DNA 칩, 겔 전기영동, 모세관 전기영동, 방사선 측정, 형광 측정 또는 인광 측정을 통해 수행될 수 있으나, 이에 제한되지 않는다. 본 발명의 하기 실시 예에서는 겔 전기영동을 통해 수행되었다.
In the method of the present invention, the detection of the amplification product by the above method may be performed through DNA chip, gel electrophoresis, capillary electrophoresis, radiation measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. In the following examples of the present invention was carried out via gel electrophoresis.
이하, 본 발명을 실시예 및 비교예에 의해 상세히 설명한다. 단, 하기 실시예 및 비교예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 비교예에 의해 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail with reference to examples and comparative examples. However, the following Examples and Comparative Examples are merely illustrative of the present invention, the contents of the present invention is not limited by the following Examples and Comparative Examples.
실시예Example 1. 미생물 균주의 준비. 1. Preparation of Microbial Strains.
본 발명에서 사용된 바실러스 세레우스와 바실러스 튜린겐시스 균주를 포함한 기타 미생물은 한국미생물보존센터(Korean Culture Center of Microorganisms, KCCM)와 생물자원센터(Biological Resource Center, BRC)에서 분양 받았으며, 그 목록은 표1에 정리하였다. 미생물의 배양은 DIFCO사의 LB(Luria-Bertani)배지와 미생물 각각의 선택배지에서 배양하였다. 바실러스 세레우스 미생물농약 분리주는 시판되는 국내산 미생물농약과 일본산 미생물농약에서 각각 4주씩 분리하여 사용하였다.Other microorganisms including Bacillus cereus and Bacillus thuringiensis strains used in the present invention were distributed at the Korean Culture Center of Microorganisms (KCCM) and the Biological Resource Center (BRC). Table 1 summarizes. The microorganisms were cultured in LIF (Luria-Bertani) medium of DIFCO company and the selective medium of each microbe. Bacillus cereus microbial pesticide isolate was used for 4 weeks from domestic and Japanese microbial pesticides.
실시예Example 2. 특이적 2. Specific 프라이머primer 셋트의Set 제작. making.
<2-1> 특이적 염기서열 확인 및 제작.<2-1> Identification and preparation of specific sequencing.
바실러스 튜린겐시스와 바실러스 세레우스를 특이적으로 검출할 수 있는 프라이머 셋트를 제작하기 위하여 NCBI(National Center for Biotechnology Information)의 GenBank와 EBI(European Bioinformatics Institute)의 alignment program을 통해 염기서열을 비교 분석한 후 바실러스 세레우스와 바실러스 튜린겐시스에 대하여 공통으로 특이적인 groEL 유전자를 target으로 선택하여 258145번~258320번 염기서열로 이루어진 176bp의 단편을 증폭하는 F1gro와 R1gro의 primer set를 제작하였다. 그 중 바실러스 튜린겐시스를 특이적으로 검출하기 위하여 곤충유충에 특이적 살충성을 나타내는 δ-endotoxin 유전자 cry를 target으로 선택하여 2964번~3101번 염기서열로 이뤄진 138bp의 단편을 증폭하는 Btc-1-NF와 Btc-1-NNR의 primer set를 제작하여 사용하였다.In order to construct a primer set capable of specifically detecting Bacillus thuringiensis and Bacillus cereus, nucleotide sequences were compared and analyzed through the GenBank of the National Center for Biotechnology Information (NCBI) and the alignment program of the European Bioinformatics Institute (EBI). Later, we selected primers of F1gro and R1gro to amplify fragments of 176bp consisting of 258145 to 258320 sequences by selecting the specific groEL gene as a target for Bacillus cereus and Bacillus thuringiensis. In order to specifically detect Bacillus thuringiensis, Btc-1, which targets the δ-endotoxin gene cry , which shows specific insecticidal properties to insect larvae, is used to amplify a fragment of 138 bp consisting of sequences 2964 to 3101. Primer sets of -NF and Btc-1-NNR were prepared and used.
<< 비교예1Comparative Example 1 > 기존 식중독균 검출 > Detection of existing food poisoning bacteria 프라이머와의With primer 검출능 비교. Detectability comparison.
본 발명의 타깃 유전자를 선정하는데 있어서의 유용성을 검증하기 위하여 식중독균 검출을 위하여 사용되고 있는 기존의 프라이머와 같은 조건에서 PCR을 수행하여 검출능을 비교하였다.In order to verify the usefulness in selecting a target gene of the present invention, PCR was performed under the same conditions as the existing primers used for the detection of food poisoning bacteria, and the detection performance was compared.
기존에 바실러스 세레우스와 같은 식중독균을 검출하기 위하여 사용되고 있는 프라이머는 bceT, gyrB를 target gene으로 사용하고 있다. 하기 실시예<3-2>에서 제시하고 있는 PCR 수행방법을 통하여 검출능을 분석하였으며 그 결과를 표2에 도시하였다. The primer used to detect food poisoning bacteria such as Bacillus cereus is using bceT and gyrB as target genes. The detection performance was analyzed by the PCR method shown in Example <3-2> and the results are shown in Table 2.
기존의 프라이머 검출능과 비교한 결과 본 발명에서 선택된 타깃 유전자의 경우 기존의 타깃 유전자를 이용하는 것과 비교하여 더 뛰어난 검출능을 보임을 확인하였다.
As a result of comparing with the existing primer detection ability, it was confirmed that the target gene selected in the present invention showed more excellent detection ability compared with the use of the existing target gene.
<2-2> F1gro/R1gro 와 Btc-1-NF/Btc-1-NNR primer set의 특이성 확인<2-2> Specificity of F1gro / R1gro and Btc-1-NF / Btc-1-NNR primer set
F1gro/R1gro primer set를 사용하여 바실러스 튜린겐시스 및 바실러스 세레우스 균주와 기타미생물을 포함한 27주를 대상으로 Thermal Cycler(Thermo Electron Co., USA)와 Taq DNA Polymerase (Solgent Co., Ltd, South Korea)를 사용하여 conventional PCR을 수행하였다. 0.2 ㎖ tube에 template DNA 1 ㎕(200 ng/㎕), 10x Taq buffer 2.5 ㎕, dNTP 0.5 ㎕(10 mM), Taq polymerase 0.125 ㎕(5 unit/㎕), 서열번호 2와 서열번호 3의 프라이머 셋트를 각각 10 pmol로 제작 후 1 ㎕씩 넣고 총 부피가 25 ㎕이 되도록 멸균 증류수를 첨가하였다. Thermal Cycler (Thermo Electron Co., USA) and Taq DNA Polymerase (Solgent Co., Ltd, South Korea) for 27 strains containing Bacillus thuringiensis and Bacillus cereus strains and other microorganisms using the F1gro / R1gro primer set. ) Was performed using conventional PCR. 1 μl (200 ng / μl) template DNA, 2.5 μl 10x Taq buffer, 0.5 μl dNTP (10 mM), 0.125 μl Taq polymerase (5 unit / μl), SEQ ID NO: 2 and SEQ ID NO: 3 in 0.2
PCR 온도 조건은 95℃에서 2분간 실시 후 95℃ 20초, 61℃ 20초, 72℃ 20초를 1 cycle로 하여 총 30 cycle을 진행한 다음 72℃에서 2분간 진행하였다. Btc-1-NF/Btc-1-NNR primer set의 특이성은 서열번호 5와 서열번호 6을 이용하여 위와 같은 조성으로 reaction mixture 만든 후 동일한 PCR온도 조건으로 진행 하여 확인하였다.PCR temperature conditions were carried out for 2 minutes at 95
증폭된 DNA는 2% 아가로오스 겔 전기영동(Mupid-2 plus, Advance, Japan)을 이용 하여 확인하였다. 2% 아가로오스 겔은 running buffer인 TAE buffer(40 mM Tris-acetate, 1 mM EDTA, pH 8.0)에 2% 아가로오스를 녹인 후, Ethidium bromide(10 mg/ml)를 60 ㎕ 첨가 한 뒤 상온에서 굳혔다. 6x loading buffer(40% sucrose, 0.25% bromophenol blue, 0.25% xylencyanol)와 DNA를 혼합하여 갤에 주입하였다. 125 V에서 25분간 전기영동 후 UV를 이용하여 증폭산물을 확인한 결과 F1gro/R1gro 프라이머 셋트는 바실러스 세레우스균과 바실러스 튜린겐시스에서 모두 증폭산물을 만들어냈고, Btc-1-NF/Btc-1-NNR 프라이머 셋트는 주요 미생물농약균주인 바실러스 튜린겐시스에만 특이적으로 반응 하여 해당 크기의 PCR 증폭산물을 만들어내는 것을 확인하였다.
Amplified DNA was confirmed using 2% agarose gel electrophoresis (Mupid-2 plus, Advance, Japan). 2% agarose gel is dissolved in 2% agarose in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0), which is a running buffer, and 60 μl of Ethidium bromide (10 mg / ml) is added. Hardened at room temperature. 6x loading buffer (40% sucrose, 0.25% bromophenol blue, 0.25% xylencyanol) and DNA were mixed and injected into the gal. After 25 minutes of electrophoresis at 125 V, the amplification product was confirmed by UV. The F1gro / R1gro primer set produced amplification products in both Bacillus cereus and Bacillus thuringiensis. Btc-1-NF / Btc-1- The NNR primer set specifically reacted only with Bacillus thuringiensis, a major microbial pesticide strain, to produce PCR amplification products of the corresponding size.
<< 실시예Example 3> 3> MultiplexMultiplex realreal -- timetime PCRPCR 을 통한 through 미생물농약균주Microbial pesticide strains 바실러스Bacillus 튜린겐시스와Turingensis and 식중독균 Food poisoning bacteria 바실러스Bacillus 세레우스의 구분검출 Detection of Sereus
<3-1> 프로브의 제작.<3-1> Probe Preparation.
멀티플렉스 리얼타임 PCR을 이용한 시스템을 구현하기 위하여 서열번호 1과 서열번호 4에서 미생물농약균주 바실러스 튜린겐시스와 식중독균 바실러스 세레우스에 공통으로 특이적인 프로브와 바실러스 튜린겐시스에만 특이적인 프로브, 2가지를 제작하였다. δ-endotoxin 유전자 cry를 타깃으로 하는 프로브의 리포터로는 FAM(emission wavelength 520 nm)을 quencher로는 BHQ1(emission wavelength 534 nm)을 사용하였고 groEL유전자를 target으로 하는 probe의 reporter로는 HEX(emission wavelength 553 nm)를 quencher로는 BHQ2(emission wavelength 579 nm)를 선택하여 제작하였다. In order to implement a system using multiplex real-time PCR, a probe specific to both the bactericidal bacterium Bacillus thuringiensis and the food poisoning bacillus cereus in SEQ ID NO: 1 and SEQ ID NO: 4 and a probe specific to Bacillus thuringiensis only Was produced. The FAM (emission wavelength 520 nm) was used as a reporter for probes targeting the δ-endotoxin gene cry , and the emission wavelength 534 nm ( BHQ1 ) was used as the quencher, and the HEX (emission wavelength 553 nm) was used as a reporter for probes targeting the groEL gene. ) Was produced by selecting BHQ2 (emission wavelength 579 nm) as the quencher.
<3-2> 멀티플렉스 리얼타임 PCR 분석.<3-2> Multiplex Real-Time PCR Analysis.
멀티플렉스 리얼타임 PCR분석은 Applied Biosystems사의 StepOneTM 리얼타임 PCR 시스템을 사용하여 수행하였다. 0.2㎖ PCR strip tube에 2 x master mix(Universal PCR Master Mix, ABI, USA) 10㎕, 서열번호 2, 3의 primer set를 각각 1㎕(10pmol), 서열번호 5, 6의 primer set를 각각 1㎕(2.5pmol), 서열번호 7의 probe 1㎕(2.5pmol), 서열번호 8의 probe 1㎕(10pmol), template DNA 1㎕(200ng/㎕)를 넣은 후 최종부피가 20㎕가 되도록 멸균증류수를 첨가하여 반응액을 제조하였다. Multiplex real-time PCR analysis was performed using a StepOne ™ real-time PCR system from Applied Biosystems. 10 μl of 2 x master mix (Universal PCR Master Mix, ABI, USA), 1 μl (10 pmol) of primer sets of SEQ ID NOS: 2, 3, and 1 primer set of SEQ ID NOs: 5, 6, respectively, in a 0.2 ml PCR strip tube. Sterile distilled water was added so that the final volume was 20 μl after adding ㎕ (2.5 pmol), 1 ㎕ (2.5 pmol) of probe of SEQ ID NO. 7, 1 ((10 pmol) of probe of SEQ ID NO. Was added to prepare a reaction solution.
멀티플렉스 리얼타임 PCR 조건은 50℃에서 2분, 95℃ 2분 반응 후 95℃ 30초, 55℃ 50초를 1 cycle로 하여 총 40 cycle을 수행하였다.
The multiplex real-time PCR conditions were performed for 40 minutes with 2 cycles at 50 ° C, 95 ° C for 2 minutes, 95 °
<3-3> 검출 한계 조사.<3-3> Detection limit investigation.
multiplex real-time PCR 분석법에 의해 미생물농약균주 바실러스 튜린겐시스와 식중독균 바실러스 세레우스가 감지되는 최저농도를 확인하기 위하여 본 발명에서 제작한 primer set와 probe를 이용하여 검출한계를 조사하였다. 미생물농약균주 바실러스 튜린겐시스 ATCC13366과 식중독균 바실러스 세레우스 ATCC21772를 배양한 후 스펙트로미터를 이용하여 배양액을 적정 농도로 맞춘 다음 1 ㎖을 취해 십진희석 하였다. 희석액을 LB plate에 도말 하여 30℃에서 24시간 배양 후 생균수 측정법으로 cell 수를 측정하였고 동시에 균액 1 ㎖을 취해 DNA를 추출하였다. 생균수 측정결과 ATCC13366은 2.2×108 cfu/㎖이었고, ATCC21772는 3.2×108 cfu/㎖이었다. 추출된 DNA를 십진 희석하여 <실시예 3-2>와 동일한 방법으로 리얼 타임 PCR을 수행한 결과 ATCC13366는 2.2×101 cfu/rxn, ATCC21772는 3.2×101 cfu/rxn까지 검출이 가능하였으며, 그 결과를 도면 4,5에 나타내었다.
In order to determine the lowest concentration of microbial pesticide strains Bacillus thuringiensis and food poisoning bacillus cereus by multiplex real-time PCR, detection limits were investigated using primer sets and probes prepared in the present invention. The microbial pesticide strain Bacillus thuringiensis ATCC13366 and the food poisoning bacterium Bacillus cereus ATCC21772 were incubated, and the culture solution was adjusted to an appropriate concentration using a spectrometer. The diluted solution was plated on an LB plate and incubated at 30 ° C. for 24 hours, and cell number was measured by viable cell count. At the same time, 1 ml of the bacterial solution was taken to extract DNA. The viable cell count was AT × 13366 2.2 × 10 8 cfu / mL and ATCC21772 was 3.2 × 10 8 cfu / mL. As a result of performing real-time PCR by decimal diluting the extracted DNA, ATCC13366 was able to detect 2.2 × 10 1 cfu / rxn and ATCC21772 up to 3.2 × 10 1 cfu / rxn. The results are shown in FIGS. 4 and 5.
서열목록 전자파일 첨부Attach an electronic file to a sequence list
Claims (9)
바실러스 튜린겐시스의 cry유전자(서열번호4)의 증폭을 위한 프라이머 셋트를 포함하는 바실러스 튜린겐시스, 바실러스 세레우스 구별용 프라이머 셋트.
Primer sets for amplification of groEL (SEQ ID NO: 1) of Bacillus thuringiensis and Bacillus cereus ;
A set of primers for distinguishing Bacillus thuringiensis and Bacillus cereus, including a set of primers for amplification of the cryogenic gene of Bacillus thuringiensis (SEQ ID NO: 4).
The primer set for distinguishing Bacillus thuringiensis and Bacillus cereus according to claim 1, wherein the primer set for amplification of groEL (SEQ ID NO: 1) is a primer set of SEQ ID NOs: 2 and 3. 3.
The primer set for distinguishing Bacillus thuringiensis and Bacillus cereus according to claim 1, wherein the primer set for amplifying the cry gene (SEQ ID NO: 4) is SEQ ID NOs: 5 and 6.
The method of claim 1, wherein the primer set for amplification of groEL (SEQ ID NO: 1) is a primer set of SEQ ID NO: 2 and 2 and the primer set for amplification of the cry gene (SEQ ID NO: 4) is SEQ ID NO: 5 and 6 A set of primers for distinguishing Bacillus thuringiensis and Bacillus cereus.
A composition for distinguishing Bacillus thuringiensis, Bacillus cereus, comprising the primer set of any one of claims 1 to 4.
The composition for distinguishing Bacillus thuringiensis, Bacillus cereus, according to claim 6, wherein the composition further comprises probes of SEQ ID NOs: 7 and 8. 8.
Bacillus thuringiensis, Bacillus cereus discriminating kit comprising the composition of claim 6.
Bacillus thuringiensis and Bacillus cereus identification method using the primer set of any one of Claims 1-4.
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