KR102091282B1 - Method for Deciding Pathogen of Species-Specific Red Rot Disease Using Restriction Fragment Length Polymorphism - Google Patents
Method for Deciding Pathogen of Species-Specific Red Rot Disease Using Restriction Fragment Length Polymorphism Download PDFInfo
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Abstract
본 발명은 붉은갯병 감염 의심 시료에 대해 김 붉은갯병을 진단하고, 원인 병원균을 정확하게 판정하기 위한 종특이적 김 붉은갯병균 결정방법에 관한 것으로, 보다 상세하게는 (A) 붉은갯병 감염이 의심되는 김 시료 또는 환경해수 시료를 준비하는 단계; (B) 시료의 전체 게놈 DNA를 추출하는 단계; (C) 추출된 전체 게놈 DNA를 주형으로 하고, 소정의 프라이머 세트를 이용하여 cox2(cytochrome c oxidase subunit 2)를 증폭하는 단계; 및 (D) 증폭된 산물에 소정의 제한효소를 처리하여 증폭된 산물이 단편화되는지 여부를 확인하는 단계;를 포함하는 종특이적 김 붉은갯병균 결정방법에 관한 것이다.The present invention relates to a method for determining a species-specific Kim red-lipped bacillus for accurately diagnosing a red-reddew disease against a suspected sample of red-lite disease and to accurately determine the causative pathogen, and more specifically, (A) suspected red-lite disease infection. Preparing a seaweed sample or an environmental seawater sample; (B) extracting the entire genomic DNA of the sample; (C) amplifying cox2 (cytochrome c oxidase subunit 2) using the extracted whole genomic DNA as a template and using a predetermined primer set; And (D) treating the amplified product with a predetermined restriction enzyme to determine whether the amplified product is fragmented.
Description
본 발명은 붉은갯병 감염 의심 시료에 대해 김 붉은갯병을 진단하고, 원인 병원균을 정확하게 판정하기 위한 종특이적 김 붉은갯병균 결정방법에 관한 것이다.The present invention relates to a species-specific method for determining a species-specific red silkworm pathogen for accurately determining the causative pathogen and diagnosing red silkworm for a sample suspected of being infected with red silkworm.
김 붉은갯병(red rot disease)은 붉은갯병균이 김(Pyropia종) 엽체에 기생해 발생하는 질병으로, 대규모 발생 시 양식김 생산량의 감소와 더불어 심각한 품질저하를 가져온다. 붉은갯병균에 감염된 김은 처음에는 둥근 반점으로 시작해 나중에는 구멍이 생기고, 감염이 심할 경우 엽체가 탈락되는 등 양식김의 생산성을 저하시키거나 미관에 문제가 생겨 품질을 저하시키는 원인이 되고 있다.Red rot disease is a disease caused by parasites of red rot disease bacteria on the laver ( Pyropia species) fronds, and when large-scale outbreaks occur, the production of farmed laver is reduced and the quality is severely deteriorated. Laver infected with Rhesus spp. starts with round spots at first, and later creates holes, and if the infection is severe, the fronds are eliminated, reducing the productivity of cultured laver or causing problems in aesthetics, which is a cause of deterioration in quality.
Pythium속의 종 중에서 P. porphyrae는, P. adhaerens 및 P. chondricola와 밀접한 유연관계를 형성하고 있지만(Levesque & De Cock 2004) 이들 세 근연종은 각기 다른 숙주/기질-특이적 관계를 보여준다(마츠모토 외. 1999, 레베 & 드 콕 2004).Among the species of the genus Pythium , P. porphyrae has a close relationship with P. adhaerens and P. chondricola (Levesque & De Cock 2004), but these three closely related species show different host/substrate-specific relationships (Matsumoto et al. 1999, Revue & De Cock 2004).
Pythium porphyrae는 지금까지 김에서 붉은갯병을 일으키는 유일한 병원체로 알려져 있었으나(다카하시 외. 1977, Kim 등. 2014) 비교적 최근에 P. chondricola 역시 붉은갯병을 유발한다는 사실이 확인되었다(Lee 등 2015, 2017). 병원균은 각 종에 따라 항생제 내성 패턴이 서로 다르기 때문에, 약제 및 처치 방법이 서로 다른 경우가 많다. 따라서 병원균을 종수준별로 감별 및 동정하는 것이 필요하다. 즉, 병원균의 정확한 종 식별은 질병에 대한 과학적 해결책을 찾고 숙주-병원균의 관계를 명확히 하는 데 중요한 것이다. 이에 따라 붉은갯병 원인균인 P. porphyrae와 P. chondricola를 구별하기 위한 몇몇 기술이 개발된 바 있다. Pythium porphyrae has been known to be the only pathogen causing red mud disease in laver so far (Takahashi et al. 1977, Kim et al. 2014), but relatively recently it was confirmed that P. chondricola also induces red mud disease (Lee et al. 2015, 2017) . As pathogens have different antibiotic resistance patterns for each species, drugs and treatment methods are often different. Therefore, it is necessary to discriminate and identify pathogens by species level. In other words, the exact species identification of the pathogen is important in finding a scientific solution to the disease and clarifying the host-pathogen relationship. Accordingly, several techniques have been developed to distinguish P. porphyrae and P. chondricola , which are the causative agents of red peony disease.
등록특허 10-1618182는, cox1 (cytochrome c oxidase subunit 1) 유전자 부위의 유전정보가 두 종을 판별하는데 유용한 정보를 가지고 있다는 사실(Lee 등 2015)에 기초하여 감염 김 엽체 및 Pythium 배양균주에서 분석 가능하도록 cox1 유전자 분석용 프라이머 조합을 새로이 개발하고 이를 붉은갯병 감염 김 시료에 적용하여 종 구분이 가능하도록 한 것이다. 등록특허 10-1734847은, 붉은갯병 감염이 의심되는 김 시료 또는 환경해수 시료에서 추출된 전체 게놈 DNA로부터 Pythium 속의 cox2 유전자를 증폭하기 위한 프라이머 세트와 상기 프라이머 세트를 이용하여 김 붉은갯병의 병원균을 결정하는 방법에 관한 것이다.Registered Patent 10-1618182 is based on the fact that the genetic information of the cox1 (cytochrome c oxidase subunit 1) gene site has useful information for discriminating two species (Lee et al. 2015), which can be analyzed in infected Kim fronds and Pythium cultured strains. A new primer combination for cox1 gene analysis was developed and applied to a sample of red seaweed infected laver so that species can be distinguished. Registered Patent 10-1734847 is a primer set for amplifying the cox2 gene in Pythium from whole genomic DNA extracted from a seaweed sample or environmental seawater sample suspected of being infected with red seaweed, and using the primer set to determine pathogens of seaweed red seaweed disease It's about how to do it.
이들 방법에 의하면 두 종을 정확하게 구분하는 것이 가능하기는 하다. 그러나 시료 전체 게놈 DNA를 추출하여(1일 소요) 이를 주형으로 하고, Pythium 속의 cox1 (또는 cox2) 유전자 증폭용 프라이머 세트를 이용하여 cox1 (또는 cox2) 유전자를 증폭한 다음 정제한다(1일 소요). 이어서 증폭된 산물의 서열을 결정하고 이를 비교하여야 한다(2~3일 소요). 염기서열 결정(시컨싱)을 위해서는 고가의 설비와 많은 시간을 필요로 하기 때문에 PCR과 전기영동장치 수준의 기존 설비만을 갖추고 있는 통상의 일반실험실에서는 수행할 수 없는, 복잡하고 많은 시간이 소요되는 방법이라는 한계가 있다.According to these methods, it is possible to accurately distinguish between the two species. However, samples by extracting total genomic DNA with this template (which takes one day), and by using the Pythium genus cox1 (or cox2) primer sets for gene amplification amplifies cox1 (or cox2) gene is then purified (which takes 1 day) . Subsequently, the sequence of the amplified product should be determined and compared (takes 2-3 days). Since it requires expensive equipment and a lot of time for sequencing (sequencing), it is a complex and time-consuming method that cannot be performed in a general laboratory equipped with only existing facilities at the level of PCR and electrophoresis devices. There is a limit of that.
본 발명은 별도의 서열결정 및 유전자 비교검색 과정 없이 PCR과 전기영동장치 수준의 설비만으로 빠르고 간편하게 붉은갯병을 발생시키는 것으로 알려진 두 종 P. porphyrae와 P. chondricola를 결정하는 방법을 제공하는 것을 목적으로 한다. An object of the present invention is to provide a method for determining two species P. porphyrae and P. chondricola , known to quickly and easily generate red blood clots with only PCR and electrophoretic equipment-level equipment without separate sequencing and gene comparison and search processes. do.
또한 본 발명은 김의 기생성 질병인 붉은갯병을 예측하고 대처할 수 있도록 병원균의 신속·정확한 검출 및 결정방법을 제공하는 것을 목적으로 한다. In addition, an object of the present invention is to provide a rapid and accurate detection and determination method of pathogens so as to predict and cope with the parasitic disease of seaweed.
전술한 목적을 달성하기 위한 본 발명은 The present invention for achieving the above object
(A) 붉은갯병 감염이 의심되는 김 시료 또는 환경해수 시료를 준비하는 단계; (B) 시료의 전체 게놈 DNA를 추출하는 단계; (C) 추출된 전체 게놈 DNA를 주형으로 하고, 소정의 프라이머 세트를 이용하여 cox2(cytochrome c oxidase subunit 2)를 증폭하는 단계; 및 (D) 증폭된 산물에 소정의 제한효소를 처리하여 증폭된 산물이 단편화되는지 여부를 확인하는 단계;를 포함하는 종특이적 김 붉은갯병균 결정방법인 것을 특징으로 한다. (A) preparing a seaweed sample or an environmental seawater sample suspected of being infected with red seaweed disease; (B) extracting the whole genomic DNA of the sample; (C) using the extracted whole genomic DNA as a template, and amplifying cox2 (cytochrome c oxidase subunit 2) using a predetermined primer set; And (D) treating the amplified product with a predetermined restriction enzyme to determine whether the amplified product is fragmented or not.
이상과 같이 본 발명에 의하면 단순한 장비만으로도 붉은갯병 의심 김 시료나 환경시료로부터 붉은갯병을 빠르고 정확하게 진단할 수 있다.As described above, according to the present invention, it is possible to quickly and accurately diagnose red peony disease from suspicious laver samples or environmental samples with simple equipment.
이에 따라 김의 품질과 수확량에 막대한 영향을 끼치고 있는 붉은갯병에 대하여 병원균의 신속·정확한 동정에 의한 효과적인 전염병 방역체계를 준비할 수 있게 된다.Accordingly, it is possible to prepare an effective infectious disease quarantine system through rapid and accurate identification of pathogens for red seaweed, which has a great influence on the quality and yield of laver.
또한 본 발명에 의하면 김 양식장에서 붉은갯병 병원균인 P. chondricola와 P. porphyrae의 분포와 감염 양상을 정확하고 신뢰성있게 모니터링할 수 있어 질병의 대량 발생 전에 조기진단을 통한 선제적인 대응이 가능하게 된다.In addition, according to the present invention, the distribution and infection patterns of P. chondricola and P. porphyrae, the pathogens of red seaweed disease, can be accurately and reliably monitored in the laver farm, enabling a preemptive response through early diagnosis before mass outbreaks of diseases.
도 1은 P. porphyrae와 P. chondricola의 cox2 유전자의 염기변이 부위를 보여주는 염기서열 정렬표.
도 2는 cox2 유전자 PCR 결과물 전기영동 사진.
도 3은 cox2 PCR 산물에 대한 제한효소 처리 결과물의 전기영동 사진.1 is a sequence alignment table showing the nucleotide mutation site of the cox2 gene of P. porphyrae and P. chondricola.
Figure 2 is an electrophoresis picture of the result of cox 2 gene PCR.
Figure 3 is an electrophoresis photograph of the result of restriction enzyme treatment for the cox 2 PCR product.
이하 첨부된 도면을 참조하여 본 발명을 보다 상세히 설명한다. 그러나 첨부된 도면은 본 발명의 기술적 사상의 내용과 범위를 쉽게 설명하기 위한 예시일 뿐, 이에 의해 본 발명의 기술적 범위가 한정되거나 변경되는 것은 아니다. 이러한 예시에 기초하여 본 발명의 기술적 사상의 범위 안에서 다양한 변형과 변경이 가능함은 당업자에게는 당연할 것이다. 또한 청구범위의 구성요소에 도면부호가 병기되어 있는 경우, 이는 설명을 위한 예시적인 것일 뿐 도면부호로 구성요소를 한정하려는 의도는 아니다.Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. However, the accompanying drawings are only examples for easily explaining the content and scope of the technical idea of the present invention, thereby not limiting or changing the technical scope of the present invention. It will be obvious to those skilled in the art that various modifications and changes are possible within the scope of the technical idea of the present invention based on these examples. In addition, when reference numerals are indicated in the components of the claims, these are illustrative only, and are not intended to limit the components to the reference numerals.
전술한 바와 같이, 본 발명은 As described above, the present invention
(A) 붉은갯병 감염이 의심되는 김 시료 또는 환경해수 시료를 준비하는 단계; (A) preparing a seaweed sample or an environmental seawater sample suspected of being infected with red seaweed disease;
(B) 시료의 전체 게놈 DNA를 추출하는 단계; (B) extracting the whole genomic DNA of the sample;
(C) 추출된 전체 게놈 DNA를 주형으로 하고, 소정의 프라이머 세트를 이용하여 cox2(cytochrome c oxidase subunit 2)를 증폭하는 단계; 및 (C) using the extracted whole genomic DNA as a template, and amplifying cox2 (cytochrome c oxidase subunit 2) using a predetermined primer set; And
(D) 증폭된 산물에 소정의 제한효소를 처리하여 증폭된 산물이 단편화되는지 여부를 확인하는 단계;를 포함하는 종특이적 김 붉은갯병균 결정방법에 관한 것이다. '증폭산물의 단편화' 여부는 통상의 전기영동을 통해 밴드의 개수를 확인함으로써 간편하게 확인할 수 있다.(D) treating the amplified product with a predetermined restriction enzyme to determine whether the amplified product is fragmented or not. Whether or not'fragmentation of the amplification product' can be easily confirmed by checking the number of bands through conventional electrophoresis.
상기 단계(C)에서 상기 소정의 프라이머 세트는 P. chondricola 및 P. porphyrae의 cox2 서열 특이적인 것으로서, 예를 들면 서열번호 1 및 서열번호 2의 프라이머 세트일 수 있으나(표 1 참조), 당연히 이에 한정되는 것은 아니다. In the step (C), the predetermined primer set is specific for the cox2 sequence of P. chondricol a and P. porphyrae , and may be, for example, a primer set of SEQ ID NO: 1 and SEQ ID NO: 2 (see Table 1), of course. It is not limited thereto.
상기 단계(C)에서 증폭된 산물은, 예를 들면 두 종류의 제한효소 처리에 의해 상기 두 병원균이 구분될 수 있다. 첫째, 상기 단계(D)에서, 제한효소로 AgsI를 적용하였을 때, 증폭된 산물이 단편화되면 P. porphyrae로, 증폭된 산물이 단편화되지 않으면 P. chondricola로 결정한다. 둘째, 상기 단계(D)에서, 상기 제한효소는 KpnI이며, 이때 증폭된 산물이 단편화되면 P. chondricola로, 증폭된 산물이 단편화되지 않으면 P. porphyrae로 결정한다.In the product amplified in step (C), the two pathogens can be distinguished by, for example, treatment with two types of restriction enzymes. First, if in the above step (D), when applying AgsI with restriction enzyme, when the amplified products into fragments P. porphyrae, the amplified products are fragmented decides P. chondricola. Second , in the step (D), the restriction enzyme is KpnI. At this time, when the amplified product is fragmented, it is determined as P. chondricola , and when the amplified product is not fragmented, it is determined as P. porphyrae.
이상과 같은 본 발명에 의하면, ① 증폭된 산물을 정제할 필요 없이 바로 제한효소를 직접 처리할 수 있으며, ② 염기서열 결정이 필요 없기 때문에 종래 알려진 종 구분(결정) 방법에 비해 시간과 비용을 대폭 절감할 수 있는 것이다.According to the present invention as described above, ① it is possible to directly process the restriction enzyme without the need to purify the amplified product, and ② significantly reduce the time and cost compared to the conventionally known species classification (determination) method because it does not require the base sequence determination. It can be saved.
[실시예] [Example]
1. 프라이머 세트의 선정1. Selection of primer set
김 및 P. porphyrae와 P. chondricola의 cox2 유전자를 분석하여 김에는 특이성이 없으면서 P. porphyrae와 P. chondricola에는 특이성이 있는 전술한 염기서열 1, 2의 프라이머 세트를 선정하였다. '염기서열 1, 2의 프라이머 세트'가 아니더라도 '김에는 특이성이 없으면서 P. porphyrae와 P. chondricola에는 특이성이 있는 프라이머 세트'가 적용될 수 있을 것이다.Incidentally it analyzes the cox2 gene of Kim and P. and P. porphyrae chondricola is eopeumyeonseo specificity porphyrae P. and P. chondricola has been selected for the above-described base sequence of the
2. DNA 추출 및 PCR 산물 획득2. DNA extraction and PCR product acquisition
(1) 하기 표 2와 같은 4종의 균주로부터 통상의 방법에 따라 각각의 게놈 DNA를 추출하였다.(1) Each genomic DNA was extracted from the four strains shown in Table 2 according to a conventional method.
(2) 추출된 게놈 DNA를 주형으로 하고 염기서열 1, 2의 프라이머 세트를 이용하여 P. porphyrae와 P. chondricola 각각의 cox2 유전자 PCR 산물을 얻었다. 두 균주 증폭 산물의 크기는 415 bp로 동일하였다(도 2). 도면에서 1~4는 표 3의 시료번호와 같다(이하 동일).(2) Using the extracted genomic DNA as a template, PCR products of the cox2 genes of P. porphyrae and P. chondricola were obtained using primer sets of
3. 제한효소 처리 및 결과물의 전기영동3. Restriction enzyme treatment and electrophoresis of the result
1 × 반응완충액, 1~5 units의 전술한 제한효소와 PCR 산물 2 ㎕를 혼합하여 총 10 ㎕이 되도록 하고(AgsI 제한효소의 경우 1㎎/㎖ BSA 1 ㎕ 추가함) 이를 37℃에서 30분~1시간 반응시켰다. 반응 결과물 3 ㎕를 1~1.4% 아가로즈 젤에 20분간 전기영동을 실시한 후 전기영동상의 밴드패턴을 확인하였다.1 × reaction buffer,
4. 결과 분석4. Analysis of results
KpnI 제한효소에 의해서 P. chondricola 증폭산물만 반응하여 약 126 bp와 289 bp인 두 개의 조각으로 전기영동상에서 구분할 수 있었다(도 3에서 (A)). 즉, KpnI 제한효소에 의해서는 P. chondricola만이 특이적으로 반응하고 P. porphyrae는 반응하지 않았다. Only the P. chondricola amplification product was reacted by KpnI restriction enzyme, and the two fragments of about 126 bp and 289 bp could be distinguished on the electrophoresis image (FIG. 3 (A)). That is, only P. chondricola reacted specifically by KpnI restriction enzyme, but not P. porphyrae.
한편, 제한효소 AgsI에 의해서는 P. porphyrae 증폭산물만이 반응하여 1개의 PCR 산물이 PCR-RFLP 반응을 통하여 약 83 bp와 332 bp인 두 개의 조각으로 전기영동상에서 구분되었다(도 3에서 (B)). 즉, AgsI 제한효소에 의해서는 P. porphyrae가 특이적으로 반응하고 P. chondricola의 cox2 산물은 반응하지 않았다. 제한효소로 절단되면 Blunt end가 아니라 비대칭형인 sticky end가 되기 때문에 절편 크기를 '약'으로 표현하였다(이하 동일).On the other hand, by the restriction enzyme AgsI, only the P. porphyrae amplification product was reacted, and one PCR product was separated into two fragments of about 83 bp and 332 bp through the PCR-RFLP reaction (Fig. 3 (B)). )). In other words, P. porphyrae reacted specifically by AgsI restriction enzyme, and the cox 2 product of P. chondricola did not react. When digested with restriction enzymes, the size of the section was expressed as'about' because it became an asymmetrical sticky end instead of a Blunt end (the same hereinafter).
따라서, 두 개의 독립적인 반응이 두 종에 대하여 각각 특이적으로 작동함으로써 높은 재연성과 신뢰성을 가지고 김 붉은갯병의 병원균이 P. chondricola인지 아니면 P. porphyrae인지를 결정할 수 있었다.Therefore, since two independent reactions acted specifically for each of the two species, it was possible to determine whether the pathogens of red mud disease were P. chondricola or P. porphyrae with high reproducibility and reliability.
<110> Republic of Korea(National Fisheries Research and Development Institute) <120> Method for Deciding Pathogen of Species-Specific Red Rot Disease Using Restriction Fragment Length Polymorphism <130> P0319-132 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gatgttattt aaaacaatag ttc 23 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 taaagaagga atagcccaa 19 <110> Republic of Korea (National Fisheries Research and Development Institute) <120> Method for Deciding Pathogen of Species-Specific Red Rot Disease Using Restriction Fragment Length Polymorphism <130> P0319-132 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gatgttattt aaaacaatag ttc 23 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 taaagaagga atagcccaa 19
Claims (3)
(B) 시료의 전체 게놈 DNA를 추출하는 단계;
(C) 추출된 전체 게놈 DNA를 주형으로 하고, 서열번호 1 및 서열번호 2의 프라이머 세트를 이용하여 cox2(cytochrome c oxidase subunit 2)를 증폭하는 단계;
(D) 증폭된 산물에 제한효소 AgsI을 처리하여 증폭된 산물이 단편화되면 P. porphyrae로, 증폭된 산물이 단편화되지 않으면 P. chondricola로 결정하는 것을 특징으로 하는 김 붉은갯병균 결정방법.
(A) preparing a seaweed sample or an environmental seawater sample suspected of being infected with red cane disease;
(B) extracting the entire genomic DNA of the sample;
(C) amplifying cox2 (cytochrome c oxidase subunit 2) using the extracted whole genomic DNA as a template and using the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2;
(D) A method for determining Kim's red pathogen, characterized in that the amplified product is determined by P. porphyrae when the amplified product is fragmented by treatment with the restriction enzyme AgsI, and by P. chondricola when the amplified product is not fragmented.
(B) 시료의 전체 게놈 DNA를 추출하는 단계;
(C) 추출된 전체 게놈 DNA를 주형으로 하고, 서열번호 1 및 서열번호 2의 프라이머 세트를 이용하여 cox2(cytochrome c oxidase subunit 2)를 증폭하는 단계;
(D) 증폭된 산물에 제한효소 KpnI을 처리하여 증폭된 산물이 단편화되면 P. chondricola로, 증폭된 산물이 단편화되지 않으면 P. porphyrae로 결정하는 것을 특징으로 하는 김 붉은갯병균 결정방법.(A) preparing a seaweed sample or an environmental seawater sample suspected of being infected with red cane disease;
(B) extracting the entire genomic DNA of the sample;
(C) amplifying cox2 (cytochrome c oxidase subunit 2) using the extracted whole genomic DNA as a template and using the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2;
(D) A method for determining Kim's red rot disease, characterized in that the amplified product is determined by P. chondricola when the amplified product is fragmented by treatment with the restriction enzyme KpnI, and by P. porphyrae when the amplified product is not fragmented.
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