KR102674008B1 - Primer set for the detection of saxitoxin biosynthesis gene - Google Patents
Primer set for the detection of saxitoxin biosynthesis gene Download PDFInfo
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- KR102674008B1 KR102674008B1 KR1020230033369A KR20230033369A KR102674008B1 KR 102674008 B1 KR102674008 B1 KR 102674008B1 KR 1020230033369 A KR1020230033369 A KR 1020230033369A KR 20230033369 A KR20230033369 A KR 20230033369A KR 102674008 B1 KR102674008 B1 KR 102674008B1
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 37
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q2531/00—Reactions of nucleic acids characterised by
- C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
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- C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
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- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
본 발명은 삭시톡신 합성 유전자를 검출할 수 있는 프라이머 세트와 이를 이용한 조성물 또는 방법에 관한 것이다. 본 발명은 열대성 침입 남조류 및 관련 독소 발생 잠재성을 분석할 수 있는 민감하고 구체적이며 신속한 프로세스를 제공하고 조기에 강력한 검출을 통해 적절한 수질 관리에 기여할 수 있다.The present invention relates to a primer set capable of detecting saxitoxin synthesis genes and a composition or method using the same. The present invention provides a sensitive, specific, and rapid process for analyzing tropical invasive blue-green algae and associated toxin generation potential and can contribute to appropriate water quality management through early and robust detection.
Description
본 발명은 삭시톡신 합성 유전자 검출용 프라이머 세트 및 이를 포함하는 조성물 또는 검출방법에 관한 것이다.The present invention relates to a primer set for detecting saxitoxin synthetic genes and a composition or detection method containing the same.
지구온난화로 대표되는 기후변화는 수생태계에 부정적인 영향을 미치며, 특히 유해 남조류가 대량 증식하여 독소와 취기 물질 등의 유해물질의 생산으로 수자원 관리에 장애를 유발하는 녹조현상(Harmful Cyanobacteria Blooms, cyanoHABs)의 규모와 빈도수를 증가시킬 가능성이 있는 것으로 보고되었다(Howard and Easthope, 2002; Komatsu et al., 2007). 대표적인 cyanoHABs 원인 남조류는 genus Microcystis, Dolichospermum, Aphanizomenon (Howard and Easthope, 2002; Sotero-Santos et al., 2008; Walker, 2014)으로 전 세계의 많은 호수와 하천에서 녹조현상과 이에 따른 피해사례가 보고되고 있다(Watson, 2004; Van Apeldoorn et al., 2007;, Paerl and Otten, 2013). 또한, 이러한 녹조현상의 원인 남조류 외에 기후변화로 인해 열대지방에서 최초 보고된 Order Nostocales의 남조류가 다른 북미, 유럽, 동북아시아 등 다른 온대지방으로 확산되고 있으며(Briand et al., 2004; Cires and Ballot, 2016; Antunes et al., 2015), 이러한 남조류를 invasive 또는 exotic cyanobacteria라 한다. 열대성 침입 남조류는 Cylindrospermopsis 속을 포함한 Sphaerospermopsis, Cuspidothrix 및 Chrysosporum 속이 있으며 (Neilan et al., 2003; Hamilton et al., 2005; Stuken et al., 2006; Kastovsky et al., 2010; Zapomelova et al., 2012; Bolius et al., 2019), 사람과 동물에 유해한 saxitoxin, cylindrospermopsin 및 anatoxin-a와 같은 독소를 생산하는 것으로 보고되었다(Sivonen, 2009; Cires and Ballot, 2016). Climate change, represented by global warming, has a negative impact on the aquatic ecosystem, especially the green algae phenomenon (Harmful Cyanobacteria Blooms, cyanoHABs), which causes problems in water resource management by producing harmful substances such as toxins and odors through the massive proliferation of harmful blue-green algae. It has been reported that there is a possibility of increasing the size and frequency of (Howard and Easthope, 2002; Komatsu et al., 2007). The representative blue-green algae that cause cyanoHABs are the genus Microcystis, Dolichospermum, and Aphanizomenon (Howard and Easthope, 2002; Sotero-Santos et al., 2008; Walker, 2014). Green algae and subsequent damage cases have been reported in many lakes and rivers around the world. (Watson, 2004; Van Apeldoorn et al., 2007;, Paerl and Otten, 2013). In addition, in addition to blue-green algae, which are the cause of this green algae phenomenon, due to climate change, blue-green algae of the Order Nostocales, which were first reported in tropical regions, are spreading to other temperate regions such as North America, Europe, and Northeast Asia (Briand et al., 2004; Cires and Ballot) , 2016; Antunes et al., 2015), these blue-green algae are called invasive or exotic cyanobacteria. Tropical invasive cyanobacteria include the genera Sphaerospermopsis, Cuspidothrix and Chrysosporum , including C ylindrospermopsis (Neilan et al., 2003; Hamilton et al., 2005; Stuken et al., 2006; Kastovsky et al., 2010; Zapomelova et al., 2012; Bolius et al., 2019), and has been reported to produce toxins such as saxitoxin, cylindrospermopsin, and anatoxin-a, which are harmful to humans and animals (Sivonen, 2009; Cires and Ballot, 2016).
최근에는 아시아권 국가인 중국, 일본에서도 이들 열대성 침입 남조류의 출현이 확인되었으며(Lei et al., 2014; Jiang et al., 2015; Hodoki et al., 2013; Chonudomkul et al., 2004), 한국의 경우 한강수계에서 Cylindrospermopsis (Raphidiopsis) raciborskii (Jeong et al., 2019)와 Sphaerospermopsis aphanizomenoides (Song and Lee, 2015)의 출현이 보고되었다. 본 연구대상지인 낙동강에서는 C. raciborskii, S. aphanizomenoides, S. renifosmis, Cuspidothrix issatchenkoi 총 4종의 출현과 24 strains이 분리배양되었다(Kim et al., 2020). 특히 낙동강에서 분리된 C. issatschenkoi의 2 strains에서 anatoxin-a 합성 유전자인 anaF와 ELISA 분석을 통해 anatoxin-a의 생산이 확인되었다. 이처럼 독소생산의 잠재성이 있고 전 세계적 확산추세인 열대성 침입 남조류는 수자원 관리를 위해 지속적인 모니터링이 필요하지만, Aphanizomenon, Dolichospermum 등 사상성남조류(nostocalean cyanobacteria)와 형태적으로 유사하여 현미경 검경을 통한 분석에 어려움이 있다(Komarek, 2013; Kim et al., 2020). Cylindrospermopsis raciborskii의 경우 종 특이적(species-specific) primer가 개발되었으며(Wilson et al., 2000), real-time PCR 등의 분석을 통한 분자생물학적 모니터링 연구가 수행되기도 하였다(Orr et al., 2010; Chiu et al., 2017). 하지만, 대부분의 열대성 침입 남조류는 현미경 분석을 이용한 형태적 분석을 수행하거나(Budzynska and Goldyn, 2017; Bolius et al., 2019; Jovanovic et al., 2015), 독소 유전자 중심의 연구가 진행되었으며(Chiu et al., 2017; Cordeiro et al., 2021; Podduturi et al., 2021), 최근에 들어서야 열대성 침입 남조류 5속(Cuspidothrix, Cylindrospermopsis, Sphaerospermopsis, Raphidiopsis, Chrysosperum)을 검출 가능한 속 특이적(genus-specific) primer가 개발되었다(Kim et al., 2021). 속 특이적 primer는 이형세포(heterocyte) 및 휴면포자(akinete)와 같은 분화세포가 발달하지 않을 시 현미경을 이용한 형태적 분석이 어려운 경우 효과적으로 열대성 침입 남조류를 검출할 수 있으며, Wilson et al.,(2000)이 개발한 Cylindrospermopsis raciborskii의 종 특이적 primer가 Raphidiopsis 속을 구분하지 못하는 단점까지 보완하였다. Recently, the appearance of these tropical invasive blue-green algae has been confirmed in Asian countries such as China and Japan (Lei et al., 2014; Jiang et al., 2015; Hodoki et al., 2013; Chonudomkul et al., 2004), and in Korea. In this case, the occurrence of Cylindrospermopsis ( Raphidiopsis ) raciborskii (Jeong et al., 2019) and Sphaerospermopsis aphanizomenoides (Song and Lee, 2015) was reported in the Han River water system. In the Nakdong River, the subject of this study, a total of four species appeared : C. raciborskii, S. aphanizomenoides, S. renifosmis, and Cuspidothrix issatchenkoi. 24 strains were isolated and cultured (Kim et al., 2020). In particular, the production of anatoxin-a was confirmed through ELISA analysis and anaF , an anatoxin-a synthesis gene, in two strains of C. issatschenkoi isolated from the Nakdong River. Tropical invasive blue-green algae, which have the potential to produce toxins and are spreading globally, require continuous monitoring for water resource management, but are morphologically similar to nostocalean cyanobacteria such as Aphanizomenon and Dolichospermum , so they cannot be analyzed through a microscope. There are difficulties (Komarek, 2013; Kim et al., 2020). In the case of Cylindrospermopsis raciborskii, species-specific primers were developed (Wilson et al., 2000), and molecular biological monitoring studies were conducted through analysis such as real-time PCR (Orr et al., 2010; Chiu et al., 2017). However, most tropical invasive cyanobacteria have been subjected to morphological analysis using microscopic analysis (Budzynska and Goldyn, 2017; Bolius et al., 2019; Jovanovic et al., 2015) or studies focusing on toxin genes (Chiu et al., 2017; Cordeiro et al., 2021; Podduturi et al., 2021), and only recently have five genera of tropical invasive blue-green algae ( Cuspidothrix, Cylindrospermopsis, Sphaerospermopsis, Raphidiopsis, and Chrysosperum ) been detected in a genus-specific manner. ) primer was developed (Kim et al., 2021). Genus-specific primers can effectively detect tropical invasive cyanobacteria when morphological analysis using a microscope is difficult when differentiated cells such as heterocytes and akinetes do not develop, and Wilson et al., ( The species-specific primer for Cylindrospermopsis raciborskii developed by (2000) even complemented the shortcoming of not being able to distinguish between the Raphidiopsis genus.
열대성 침입 남조류의 모니터링은 대상 조류의 출현 여부와 함께 독소 발생의 가능성을 확인해야 하며, 기존에 개발된 독소검출 primers(Kellmann et al., 2006; Mihali et al., 2008; Gkelis et al, 2014; Ballot et al., 2018; Kim et al., 2020)와 속 특이적 primers를 기반으로 유전자 정량분석을 수행한다면, 열대성 침입 남조류에 의한 독소 발생 가능성을 효과적으로 파악할 수 있을 것이다. Monitoring of tropical invasive blue-green algae requires checking the appearance of the target algae and the possibility of toxin generation, using previously developed toxin detection primers (Kellmann et al., 2006; Mihali et al., 2008; Gkelis et al, 2014; By performing quantitative gene analysis based on Ballot et al., 2018; Kim et al., 2020) and genus-specific primers, the possibility of toxins caused by tropical invasive blue-green algae can be effectively identified.
따라서, 본 연구에서는 열대성 침입 남조류를 대상으로 삭시톡신 합성 유전자(sxtI) 프라이머에 기반한 유전자 정량분석 기법을 개발하고, 낙동강 8개 지점의 열대성 침입 남조류와 관련 독소유전자 보유 남조류의 출현 경향을 분석함으로써 기후변화에 따른 열대성 침입 남조류의 발생 및 독소 발생 가능성을 분석하고자 하였다. Therefore, in this study, we developed a gene quantitative analysis technique based on saxitoxin synthesis gene ( sxtI ) primers targeting tropical invasive blue-green algae, and analyzed the appearance trends of tropical invasive blue-green algae and blue-green algae carrying related toxin genes in eight locations in the Nakdong River to predict climate change. We sought to analyze the occurrence of tropical invasive blue-green algae and the possibility of toxin generation due to changes.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 삭시톡신 합성 유전자 검출용 프라이머 세트, 삭시톡신 합성 유전자 검출용 조성물 및 이를 이용한 삭시톡신 합성 유전자 검출 방법 또는 수중 독소생산 예측 방법을 확인하여 본 발명을 완성하였다.The present invention was developed in response to the above-mentioned needs, and the present inventors identified a primer set for detecting saxitoxin synthesis genes, a composition for detecting saxitoxin synthesis genes, and a method for detecting saxitoxin synthesis genes or a method for predicting toxin production in water using the same. The present invention has been completed.
상기 과제를 해결하기 위하여, 본 발명은 서열번호 1 및 서열번호 2의 프라이머; 및 서열번호 3의 프로브를 포함하는 삭시톡신 합성 유전자 검출용 프라이머 세트를 제공한다. In order to solve the above problem, the present invention provides primers of SEQ ID NO: 1 and SEQ ID NO: 2; and a primer set for detecting saxitoxin synthesis genes including the probe of SEQ ID NO: 3.
본 발명의 일 예에서, 상기 프라이머 세트는 실시간 중합효소 연쇄반응(real-time PCR), 중첩 중합효소 연쇄반응(nested PCR), 액적 디지털 중합효소 연쇄반응(droplet digital PCR) 또는 재조합효소 중합효소 증폭(recombinase polymerase amplification)에 사용하기 위한 것일 수 있다. In one example of the invention, the primer set is used for real-time polymerase chain reaction (real-time PCR), nested polymerase chain reaction (nested PCR), droplet digital polymerase chain reaction (droplet digital PCR) or recombinase polymerase amplification. It may be for use in (recombinase polymerase amplification).
본 발명의 다른 예에서, 상기 프라이머 세트는 프로브를 포함하는 것일 수 있고, 상기 프로브는 5' 말단에 형광단이 표지되고, 3' 말단에 소광체가 표지된 것이고, 상기 형광단은 플루오레세인(fluorescein), 6-카르복시플루오레세인(FAM, 6-carboxyfluorescein), 헥사클로로-6-카르복시플루오레세인(HEX, hexachloro-6-carboxyfluorescein), 테트라클로로-6-카르복시플루오레세인(TET, tetrachloro-6-carboxyfluorescein), 2-클로로-7-페닐-1,4-디클로로-6-카르복시플루오레세인(VIC, 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein), 2,7-디메톡시-4,5-디클로로-6-카르복시플루오레세인(JOE, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein), 5-((2-아미노에틸)아미노)나프탈렌-1-술폰산(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), 쿠마린(coumarin) 및 쿠마린 유도체, 시아닌-5(Cy5, Cyanine-5), 루시퍼 옐로우(lucifer yellow), 텍사스 레드(texas red), 테트라메틸로다민(tetramethylrhodamine), 야키마 옐로우(YG, Yakima Yellow), 및 칼 플루오르 레드 610(CFR, Cal Fluor Red 610)으로 이루어진 군으로부터 선택되는 하나 이상이며, 상기 소광체는 테트라메틸로다민(TAMRA, tetramethylrhodamine), 4-(4-디메틸아미노페닐아조)벤조산(4-(4-dimethylaminophenylazo)benzoic acid), 4-디메틸아미노페닐아조페닐-4-말레이미드(4-dimethylaminophenylazophenyl-4-maleimide), 카르복시테트라메틸로다민(carboxytetramethylrhodamine) 및 BHQ 염료(BHQ dyes)로 이루어진 군으로부터 선택되는 하나 이상일 수 있다. In another example of the present invention, the primer set may include a probe, wherein the probe is labeled with a fluorophore at the 5' end and a quencher at the 3' end, and the fluorophore is fluorescein ( fluorescein), 6-carboxyfluorescein (FAM, 6-carboxyfluorescein), hexachloro-6-carboxyfluorescein (HEX, hexachloro-6-carboxyfluorescein), tetrachloro-6-carboxyfluorescein (TET, tetrachloro- 6-carboxyfluorescein), 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC, 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein), 2,7 -Dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein), 5-((2-aminoethyl)amino)naphthalene-1 -Sulfonic acid (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), coumarin and coumarin derivatives, cyanine-5 (Cy5, Cyanine-5), lucifer yellow, Texas red ( texas red), tetramethylrhodamine, Yakima Yellow (YG), and Cal Fluor Red 610 (CFR), and the quencher is tetramethylrhodamine. Methyrhodamine (TAMRA, tetramethylrhodamine), 4-(4-dimethylaminophenylazo)benzoic acid, 4-dimethylaminophenylazophenyl-4-maleimide (4-dimethylaminophenylazophenyl-4) -maleimide), carboxytetramethylrhodamine, and BHQ dyes.
상기 프라이머 또는 프로브의 농도는 실험 조건에 따라 통상의 기술자가 다양하게 선택하여 사용할 수 있고, 바람직하게는 각각 1 내지 1000 nM일 수 있고, 더욱 바람직하게는 각각 100 내지 500 nM일 수 있으나, 이에 한정되는 것은 아니다.The concentration of the primer or probe can be selected in various ways by a person skilled in the art depending on the experimental conditions, and may preferably be 1 to 1000 nM, and more preferably 100 to 500 nM, but is limited thereto. It doesn't work.
상기 실시간 중합효소 연쇄반응을 실시하는 단계는 30 내지 50 사이클, 30 내지 46 사이클, 30 내지 44 사이클, 33 내지 50 사이클, 33 내지 46 사이클, 33 내지 44 사이클, 36 내지 50 사이클, 36 내지 46 사이클, 36 내지 44 사이클, 38 내지 50 사이클 또는 38 내지 46 사이클, 예를 들어, 38 내지 44 사이클 조건으로 수행되는 것일 수 있으나, 이에 한정되는 것은 아니다.The step of performing the real-time polymerase chain reaction is 30 to 50 cycles, 30 to 46 cycles, 30 to 44 cycles, 33 to 50 cycles, 33 to 46 cycles, 33 to 44 cycles, 36 to 50 cycles, and 36 to 46 cycles. , 36 to 44 cycles, 38 to 50 cycles, or 38 to 46 cycles, for example, may be performed under 38 to 44 cycle conditions, but are not limited thereto.
본 발명은 상기 프라이머 세트를 포함하는 삭시톡신 합성 유전자 검출용 조성물을 제공한다.The present invention provides a composition for detecting saxitoxin synthesis genes comprising the above primer set.
또한, 본 발명은 상기 프라이머 세트를 포함하는 수중 독소생산 예측용 조성물을 제공한다.Additionally, the present invention provides a composition for predicting toxin production in water comprising the primer set.
본 발명의 다른 예에서, 검출용 또는 예측용 시료 또는 상기 수중은 연못, 강, 계곡, 호수, 표층수, 담수, 염수, 지하수, 세류, 공정수, 공업 용수, 농업 용수 및 식수로 이루어진 군에서 선택된 것일 수 있으나, 이에 한정되는 것은 아니다. In another example of the present invention, the sample for detection or prediction or the water is selected from the group consisting of ponds, rivers, valleys, lakes, surface water, fresh water, salt water, groundwater, trickle water, process water, industrial water, agricultural water, and drinking water. It may be, but is not limited to this.
본 발명의 또 다른 예에서, 상기 독소는 삭시톡신인 것일 수 있다.In another example of the present invention, the toxin may be saxitoxin.
본 발명은 분리된 DNA 또는 RNA 시료를 준비하는 단계; 상기 프라이머 세트를 사용하여 중합효소 연쇄반응으로 증폭하는 단계; 및 증폭 결과를 획득하는 단계를 포함하는 삭시톡신 합성 유전자 검출 방법을 제공한다. The present invention includes the steps of preparing an isolated DNA or RNA sample; Amplifying by polymerase chain reaction using the primer set; And it provides a method for detecting saxitoxin synthesis genes, including the step of obtaining amplification results.
또한, 본 발명은 DNA 또는 RNA 시료를 준비하는 단계; 상기 프라이머 세트를 사용하여 중합효소 연쇄반응으로 증폭하는 단계; 및 증폭 결과를 획득하는 단계를 포함하는 수중 독소생산 예측 방법을 제공한다. Additionally, the present invention includes the steps of preparing a DNA or RNA sample; Amplifying by polymerase chain reaction using the primer set; and providing a method for predicting toxin production in water, including the step of obtaining amplification results.
본 발명의 삭시톡신 합성 유전자를 검출할 수 있는 프라이머 세트와 이를 이용한 조성물 또는 방법은 수중에서 남조류 독소 발생 잠재성을 분석할 수 있는 민감하고 구체적이며 신속한 프로세스를 제공하고 조기에 강력한 검출을 통해 적절한 수질 관리에 기여할 수 있을 것이다.The primer set capable of detecting the saxitoxin synthesis gene of the present invention and the composition or method using the same provide a sensitive, specific, and rapid process for analyzing the potential for blue-green algae toxin generation in water and ensure appropriate water quality through early and powerful detection. You will be able to contribute to management.
도 1은 본 발명의 일 구현 예에 따른 대한민국 낙동강 본류의 샘플링 위치를 나타낸 것이다.
도 2는 본 발명의 일 구현 예에 따른 Aphanizomenon NIVA-851, sxtI 유전자 카피 및 삭시톡신 농도의 관계를 나타낸 것이다.
도 3은 본 발명의 일 구현 예에 따른 낙동강에서 독소 유전자를 세포수로 변환하여 정량적 유전자 분석 결과(2020)를 나타낸 것이다. Figure 1 shows the sampling location of the main stream of the Nakdong River in Korea according to an embodiment of the present invention.
Figure 2 shows the relationship between Aphanizomenon NIVA-851, sxtI gene copy, and saxitoxin concentration according to an embodiment of the present invention.
Figure 3 shows the results of quantitative genetic analysis (2020) by converting toxin genes into cell numbers in the Nakdong River according to an embodiment of the present invention.
이하, 본 발명의 바람직한 구현 예에 대하여 상세히 설명한다. 또한, 하기의 설명에서는 구체적인 구성요소 등과 같은 많은 특정 사항들이 도시되어 있는데, 이는 본 발명의 보다 전반적인 이해를 돕기 위해서 제공된 것일 뿐 이러한 특정 사항들 없이도 본 발명이 실시될 수 있음은 이 기술분야에서 통상의 지식을 가진 자에게는 자명하다 할 것이다. 그리고, 본 발명을 설명함에 있어서, 관련된 공지 기능 혹은 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details, such as specific components, are shown, but this is provided to facilitate a more general understanding of the present invention, and it is known in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge. Additionally, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted.
<실시예 1> 열대성 침입 남조류 선정 및 독소생산 남조류 배양<Example 1> Selection of tropical invasive blue-green algae and cultivation of toxin-producing blue-green algae
유전자 정량분석 시스템 구축을 위해 열대성 침입 및 독소생산 남조류를 낙동강에서 분리하거나, 국제 조류 배양 센터에서 분양받아 확보하였다. 본 연구에서 타겟으로 하는 남조류 독소는 열대성 침입 남조류를 포함한 유해남조류가 생산하는 것으로 알려진 saxitoxin을 대상으로 선정하였다. Saxitoxin은 독소합성 유전자 sxtI 및 독소생산이 확인된 Aphanizomenon gracile (NIVA-851, Oslo, Norway) strain를 분양받아 사용하였다. 낙동강에서 분리한 열대성 침입 남조류(Kim et al., 2020)와 분양받은 독소 positive control strains는 MLA (Bolch and Blackburn, 1996)배지에서 20℃, 40 μEm2s1, L/D =14/10 hr 광조건으로 배양하였으며, 유전자 정량 분석시 대수성장기까지 증식시켜 분석에 사용하였다(표 1).To build a quantitative genetic analysis system, tropical invasive and toxin-producing blue-green algae were isolated from the Nakdong River or acquired from the International Algae Culture Center. The blue-green algae toxin targeted in this study was saxitoxin, which is known to be produced by harmful blue-green algae, including tropical invasive blue-green algae. Saxitoxin was used from an Aphanizomenon gracile (NIVA-851, Oslo, Norway) strain with a confirmed toxin synthesis gene sxtI and toxin production. Tropical invasive blue-green algae isolated from the Nakdong River (Kim et al., 2020) and toxin positive control strains were grown on MLA (Bolch and Blackburn, 1996) medium at 20°C, 40 μEm 2 s 1 , L/D = 14/10 hr. It was cultured under light conditions, and used for quantitative gene analysis by growing until the logarithmic growth phase (Table 1).
<실시예 2> Isolation of Genomic DNA<Example 2> Isolation of Genomic DNA
Genomic DNA의 추출은 위해, 배양조류 및 현장시료를 0.45μm MicronSep nitrocelluslose membrane disk(GVS Life Sciences, Findley, OH, USA)로 여과하였고(사용 전까지 -80℃에 보관), QIAGEN DNeasy Plant Mini Kit(QIAGEN, Hilden, Germany)를 사용하여 genomic DNA를 추출하였다. 유전자 추출 절차는 -80℃에서 보관한 여과지를 상온에 5분간 정치한 뒤 lysis buffer를 넣고 소니케이터를 이용하여 파쇄한 뒤 kit에서 제공한 프로토콜을 따랐다. 추출한 genomic DNA는 Infinite M200 PRO를 이용하여 농도와 순도를 측정했다. 추출한 genomic DNA는 -20℃에서 보관하였다.For extraction of genomic DNA, cultured algae and field samples were filtered through 0.45μm MicronSep nitrocelluslose membrane disk (GVS Life Sciences, Findley, OH, USA) (stored at -80°C until use), and QIAGEN DNeasy Plant Mini Kit (QIAGEN , Hilden, Germany) was used to extract genomic DNA. The gene extraction procedure followed the protocol provided in the kit by leaving the filter paper stored at -80°C at room temperature for 5 minutes, adding lysis buffer, and disrupting it using a sonicator. The concentration and purity of the extracted genomic DNA were measured using Infinite M200 PRO. Extracted genomic DNA was stored at -20°C.
<실시예 3> 유전자 정량분석을 위한 독소 특이적 primer 제작<Example 3> Production of toxin-specific primers for quantitative gene analysis
본 연구에서는 ddPCR (digital droplet PCR)을 이용하여 유전자 정량분석을 수행하였다. ddPCR은 20μL의 reaction mixture를 20,000 이상의 droplets로 나누어 증폭하고, target DNA의 증폭 여부에 따라 positive droplet과 negative droplet으로 구분하여 target DNA의 copy 수를 산출하는 방법으로 복잡한 혼합물 상태에서 대상 유전자를 정밀하게 검출할 수 있는 장점이 있으며, 최근 해양 유해조류 유전자 정량분석 (Lee et al., 2020), 정수장 미생물 연구(Kitajima et al., 2020) 및 식물 잎에서 독성남조류 검출 연구(Lee et al., 2021) 등에 사용되었다. In this study, quantitative gene analysis was performed using ddPCR (digital droplet PCR). ddPCR accurately detects the target gene in a complex mixture by amplifying 20 μL of the reaction mixture into more than 20,000 droplets and calculating the copy number of the target DNA by dividing them into positive and negative droplets depending on whether the target DNA is amplified. There is an advantage in this, and recent quantitative analysis of marine harmful algae genes (Lee et al., 2020), research on water purification plant microorganisms (Kitajima et al., 2020), and research on detection of toxic cyanobacteria in plant leaves (Lee et al., 2021) It was used, etc.
유전자 정량분석을 위한 primer의 제작은 선행연구에서 제작된 saxitoxin 독소 합성 유전자(sxtI) 검출 primers (Kellmann et al., 2006; Mihali et al., 2008; Gkelis et al, 2014; Ballot et al., 2018; Kim et al., 2020)를 기반으로 forward와 reverse primer 외에 probe가 포함된 primer set를 제작하였다. Primer set의 제작은 ddPCR 운영 매뉴얼에 따라, PCR product의 크기 250bp 이하, probe의 Tm값은 primer 쌍보다 5∼10℃ 이상 높고, probe의 시작 염기가 G가 아니어야 하고, primer와 probe간에 서로 dimer를 형성하지 않도록 상보적인 염기서열이 없는 조건을 고려하여 제작하였다(표 2).Primers for quantitative gene analysis were prepared using saxitoxin toxin synthesis gene ( sxtI ) detection primers produced in previous studies (Kellmann et al., 2006; Mihali et al., 2008; Gkelis et al., 2014; Ballot et al., 2018). Based on (Kim et al., 2020), a primer set containing probes in addition to forward and reverse primers was created. Primer set production is based on the ddPCR operation manual. The size of the PCR product must be 250bp or less, the Tm value of the probe must be 5 to 10°C higher than that of the primer pair, the starting base of the probe must not be G, and there must be a dimer between the primer and probe. It was produced considering the conditions without complementary base sequences to prevent the formation of (Table 2).
<실시예 4> ddPCR(Digital droplet PCR)을 이용한 유전자 정량분석 <Example 4> Quantitative gene analysis using ddPCR (digital droplet PCR)
T100 Thermal Cycler로 구성된 QX200 ddPCR 시스템은 단일세포에서 각 타겟 유전자 copy 수를 측정하는 장비이다(Bio-Rad Laboratories Inc., CA, USA). ddPCR 수행시 최종볼륨 20 μL로 각 반응 혼합물은 10 μL of ddPCR™ Supermix for Probe (no dUTP) (Bio-Rad Laboratories Inc., Munich, Germany), 0.9 μL each of 10 pmol of forward and reverse primer, 0.25 μL of 10 pmol of probe 및 1 μL of sample DNA로 조성하며, 멸균 증류수로 최종 볼륨을 맞춘다. 반응 혼합물은 96-well plate에 분주하여, 각각의 반응 혼합물을 Bio-Rad QX200TM Droplet Generator (Bio-Rad Lab Inc., Hercules, CA, USA)에서 약 1 nanoliter의 20,000개의 droplet으로 나눠 새로운 96-well plate로 옮겨 T100 Thermal Cycler (Bio-Rad Lab Inc., Hercules, CA, USA)에서 PCR을 진행한다. PCR 반응후 플레이트는 Droplet reader (Bio-Rad Lab Inc., Hercules, CA, USA)로 옮겨 droplet의 FAM 또는 HEX 신호를 측정하여 정량한다. 신호 분석은 QuantaSoft™ software version 1.7.4 (Bio-Rad Laboratories Inc., CA, USA)을 이용하여 수행하였으며 최소 12000 droplet 이상의 신호가 읽혔을 때만 측정값으로 사용한다(Huggett et al., 2013). 형광 신호의 threshold 값은 negative와 positive control의 신호 값에 따라 직접 설정한다. ddPCR과 관련된 모든 시약은 Bio-Rad(Bio-Rad Laboratories Inc., USA)에서 구입하였다. The QX200 ddPCR system, which consists of a T100 Thermal Cycler, is an equipment that measures the copy number of each target gene in a single cell (Bio-Rad Laboratories Inc., CA, USA). When performing ddPCR, the final volume is 20 μL, and each reaction mixture contains 10 μL of ddPCR ™ Supermix for Probe (no dUTP) (Bio-Rad Laboratories Inc., Munich, Germany), 0.9 μL each of 10 pmol of forward and reverse primer, 0.25 It is composed of μL of 10 pmol of probe and 1 μL of sample DNA, and the final volume is adjusted with sterile distilled water. The reaction mixture was dispensed into a 96-well plate, and each reaction mixture was divided into 20,000 droplets of approximately 1 nanoliter in a Bio-Rad QX200TM Droplet Generator (Bio-Rad Lab Inc., Hercules, CA, USA) and placed in a new 96-well. Transfer to a plate and perform PCR on a T100 Thermal Cycler (Bio-Rad Lab Inc., Hercules, CA, USA). After the PCR reaction, the plate is transferred to a Droplet reader (Bio-Rad Lab Inc., Hercules, CA, USA) and quantified by measuring the FAM or HEX signal of the droplet. Signal analysis was performed using QuantaSoft™ software version 1.7.4 (Bio-Rad Laboratories Inc., CA, USA), and was used as a measurement value only when a signal of at least 12000 droplets was read (Huggett et al., 2013). The threshold value of the fluorescence signal is set directly according to the signal values of negative and positive controls. All reagents related to ddPCR were purchased from Bio-Rad (Bio-Rad Laboratories Inc., USA).
<실시예 5> 독소생산 남조류의 세포당 독소 유전자 copy 수 산출<Example 5> Calculation of toxin gene copy number per cell of toxin-producing blue-green algae
독소 생산남조류의 세포당 독소 유전자 copy 수 산출은 독소생산이 확인된 strains (NIVA-851)을 배양하여 세포 밀도별로 희석하고, membrane disk에 여과하여 Genomic DNA 추출 후 독소 검출 유전자(saxitoxin: sxtI)를 대상으로 ddPCR을 수행하였다. 각 희석된 시료 15mL을 루골용액으로 고정하여 위상차 현미경으로 세포밀도를 분석하였으며, 유전자 정량분석 결과를 이용하여 최종적으로 세포당 독소 유전자 copy 수(copies cell-1)를 산정하였다.To calculate the number of toxin gene copies per cell of toxin-producing blue-green algae, strains (NIVA-851) confirmed to produce toxin were cultured, diluted by cell density, filtered on a membrane disk, extracted genomic DNA, and toxin detection gene (saxitoxin: sxtI ). ddPCR was performed on the target. 15 mL of each diluted sample was fixed with Lugol's solution and the cell density was analyzed using a phase contrast microscope, and the final toxin gene copy number per cell (copies cell -1 ) was calculated using the quantitative gene analysis results.
<실시예 6> 낙동강 8개 지점의 열대성 침입 남조류 분석<Example 6> Analysis of tropical invasive blue-green algae at eight locations in the Nakdong River
열대성 침입 남조류 모니터링은 낙동강 상류부터 하류까지 8개 보 지점(Sangju, SJ; Nakdan, ND; Gumi, GM; Chilgok, CG; Gangjeong-Goryeong, GG; Dalseong, DS; Hapcheon-Changnyeong, HC; and Changnyeong-Haman, CH)을 대상으로 3월부터 11월까지 2주 간격(6월∼9월은 주 1회)으로 보 상류 500m 지점에서 표층수 2L를 채집하여 사용하였다(도 1). 채집한 시료는 4℃를 유지하여 실험실로 이동하였고, 현미경 분석을 위한 시료는 유리 bottle에 현장수 1L를 루골용액 0.3%로 고정하고, 자연침전법으로 48시간 이상 정치 후 사이펀으로 상등액을 제거하여, 5∼8배 농축하였다. 유전자 정량분석을 위한 시료는 현장수 20 mL를 0.45 μm MicronSep nitrocelluslose membrane disk로 여과하여, Genomic DNA를 추출하였으며, 추출한 DNA는 -20℃에서 보관했다. 독소분석을 위한 시료는 50 mL conical tube에 현장수를 넣고 -20℃에서 보관하였으며, 분석 시 시료를 4℃ 냉장고에서 해동 후 초음파 파쇄기로 세포를 파쇄하여 독소 분석을 위한 각각의 tube로 이동 후, 보존용액을 넣어 처리하였다. Monitoring of tropical invasive blue-green algae was conducted at eight weir points from the upstream to the downstream of the Nakdong River (Sangju, SJ; Nakdan, ND; Gumi, GM; Chilgok, CG; Gangjeong-Goryeong, GG; Dalseong, DS; Hapcheon-Changnyeong, HC; and Changnyeong- Haman, CH), 2L of surface water was collected and used at 500m upstream of the weir at two-week intervals from March to November (once a week from June to September) (Figure 1). The collected samples were maintained at 4°C and transported to the laboratory. For samples for microscopic analysis, 1L of field water was fixed in a glass bottle with 0.3% Lugol's solution, and after standing for more than 48 hours using the natural precipitation method, the supernatant was removed with a siphon. , concentrated 5 to 8 times. For samples for quantitative genetic analysis, 20 mL of field water was filtered through a 0.45 μm MicronSep nitrocelluslose membrane disk, and genomic DNA was extracted. The extracted DNA was stored at -20°C. Samples for toxin analysis were stored at -20°C in a 50 mL conical tube with field water. Upon analysis, the sample was thawed in a 4°C refrigerator, the cells were disrupted with an ultrasonic disruptor, and then transferred to each tube for toxin analysis. Preservation solution was added and treated.
열대성 침입 남조류의 현미경을 이용한 형태적 분석은 농축된 시료 1 mL을 Sedgwick-Rafter counting chamber를 사용하여 위상차 현미경(ZEISS, Germany) 하에서 100∼400배율로 Komarek(2013)과 John et al.(2011)을 참고하여 분석하였다. 열대성 침입 남조류의 검경은 각 종의 중요 분류 key인 정단세포(terminal cell)의 특징, 이형세포(heterocyte)의 형성 유무와 위치, 휴면포자(akinete)의 발생 위치 등 명확한 종의 구분이 가능한 세포만을 검경 하였으며, 농축 비율을 환산하여 최종 1 mL 당 세포수를 산정하였다. 독소 primer를 이용한 독소 유전자 보유 남조류의 정량분석은 genomic DNA를 독소 primer를 이용하여 ddPCR을 수행하였으며, target 유전자의 copy 수는 본 연구에서 산정된 세포 당 copy 수를 이용하여 최종 1 mL 당 독소 유전자 보유 남조류의 세포수를 산정하였다. Microscopic morphological analysis of tropical invasive blue-green algae was performed using 1 mL of concentrated samples under a phase contrast microscope (ZEISS, Germany) at 100-400 magnification using a Sedgwick-Rafter counting chamber, as described by Komarek (2013) and John et al. (2011). It was analyzed with reference to . The speculum of tropical invasive blue-green algae is only used for cells that can be clearly classified into species, including the characteristics of terminal cells, the formation and location of heterocytes, and the location of dormant spores (akinete), which are important classification keys for each species. Speculum was performed, and the concentration ratio was converted to calculate the final number of cells per 1 mL. Quantitative analysis of blue-green algae possessing toxin genes using toxin primers was performed using ddPCR on genomic DNA using toxin primers. The copy number of target genes was calculated using the copy number per cell calculated in this study to determine toxin gene retention per final mL. The number of blue-green algae cells was calculated.
<실시예 7> Toxin Analysis Using ELISA Kit<Example 7> Toxin Analysis Using ELISA Kit
Saxitoxin(52255B) ELISA 키트(Eurofins Abraxis, Inc., Warminster, PA, USA)를 이용하여 배양된 남조류 주(cyanobacteria strains)와 8개 보 근처에서 채취한 물 시료의 독소 분석을 수행하였다. 분석을 위해 배양된 남조류와 물 샘플을 50mL 튜브에 넣고 얼음 위에서 초음파 처리한 다음 제조업체의 지침에 따라 ELISA를 수행했다. Infinite M200 PRO 마이크로플레이트 판독기(Tecan Austria GmbH, Grodig, Austria)를 사용하여 450nm에서 흡광도를 측정했다.Saxitoxin (52255B) ELISA kit (Eurofins Abraxis, Inc., Warminster, PA, USA) was used to analyze toxins from cultured cyanobacteria strains and water samples collected near eight reservoirs. For analysis, cultured blue-green algae and water samples were placed in 50 mL tubes, sonicated on ice, and ELISA was performed according to the manufacturer's instructions. Absorbance was measured at 450 nm using an Infinite M200 PRO microplate reader (Tecan Austria GmbH, Grodig, Austria).
<시험예 1> 독소생산 남조류의 세포당 독소 유전자 copy 수 및 독소 농도 산출<Test Example 1> Calculation of toxin gene copy number and toxin concentration per cell of toxin-producing blue-green algae
독소(saxitoxin) 유전자 copy 수 및 독소농도 측정결과, Saxitoxin를 생산하는 Aphanizomenon NIVA-851의 세포 밀도가 1,826∼14,620 cells mL-1의 범위일 때 sxtI 유전자가 4,347∼22,800 copies mL-1 범위였고, 독소농도는 0.001∼0.016 μg L-1로 나타났다(도 2). 독소 유전자는 세포수 및 독소농도와 비례적인 관계(r2=0.96 이상)를 나타냈으며, saxitoxin은 sxtI 유전자가 평균 2.1 copies cell-1, 독소는 0.001 pg cell-1로 나타났다(도 2, 표 3).As a result of measuring the saxitoxin gene copy number and toxin concentration, when the cell density of Aphanizomenon NIVA-851, which produces saxitoxin, was in the range of 1,826 to 14,620 cells mL -1 , the sxtI gene was in the range of 4,347 to 22,800 copies mL -1 , and the toxin The concentration was found to be 0.001∼0.016 μg L -1 (Figure 2). The toxin gene showed a proportional relationship (r 2 = 0.96 or more) with the number of cells and toxin concentration, and the sxtI gene for saxitoxin showed an average of 2.1 copies cell -1 and the toxin showed an average of 0.001 pg cell -1 (Figure 2, Table 3 ).
GeneTarget
Gene
(Copies Cell-1)Gene
(Copies Cell -1 )
(pg Cell-1)Toxin
(pg Cell -1 )
<시험예 2> 낙동강수계 독소유전자 보유 남조류 분석<Test Example 2> Analysis of blue-green algae carrying toxin genes in the Nakdong River system
분석결과 Saxitoxin 합성 유전자(sxtI)는 0∼101 copies mL-1의 범위이며, 세포수로 환산결과 0∼48 cells mL-1로 나타났다. 각 지점별로 7월 16일 낙단보(ND)에서 가장 현존량이 높았고, 4월∼6월까지 구미보(GM), 합천창녕보(HC), 창녕함안보(CH)에서 20 cells mL-1 이상 검출되었으며, 그 외 지점에서는 10 cells mL-1 미만으로 낮게 검출되었다(도 3).As a result of the analysis, the Saxitoxin synthesis gene ( sxtI ) ranged from 0 to 101 copies mL -1 , and when converted to cell number, it was found to be 0 to 48 cells mL -1 . At each location, the highest present volume was at Nakdanbo (ND) on July 16, and more than 20 cells mL -1 at Gumibo (GM), Hapcheon-Changnyeongbo (HC), and Changnyeong-Hamanbo (CH) from April to June. was detected, and at other points, it was detected as low as less than 10 cells mL -1 (Figure 3).
<시험예 3> ELISA kit를 이용한 독소분석<Test Example 3> Toxin analysis using ELISA kit
ELISA kit를 이용하여 낙동강수계 8개 지점의 독소 분석 결과, saxitoxin은 4월∼5월에 칠곡보(CG), 강정고령보(GG), 달성보(DS), 창녕함안보(CH)에서 0.023∼0.043 ng L-1의 범위로 미량 검출되었다(표 4). As a result of toxin analysis at eight points in the Nakdong River system using an ELISA kit, saxitoxin was 0.023 at Chilgok Weir (CG), Gangjeong Goryeong Weir (GG), Dalseong Weir (DS), and Changnyeong Haman Weir (CH) from April to May. A trace amount was detected in the range of ∼0.043 ng L -1 (Table 4).
<시험예 4> Discussion<Test Example 4> Discussion
독소검출 primer를 이용한 유전자 정량분석 결과 독소(saxitoxin)는 2 copies cell-1 수준으로 나타났으며, 독소 유전자 copy 수는 남조류 세포수 뿐만 아니라, 각 독소농도와도 비례적인 관계를 나타냈다. 하지만, 독소의 농도는 대상 조류의 성장단계나 영양염류와 수온 등 환경조건에 따라 변동될 가능성이 있기 때문에(Chiu et al., 2017; Orr et al.. 2010) 독소 유전자 정량분석 결과를 이용하여 정확한 독소농도를 산출할 수 없지만, 독소 발생의 잠재성을 판단하는 자료로 활용 가능할 것으로 판단된다. As a result of quantitative gene analysis using toxin detection primers, the toxin (saxitoxin) was found to be at the level of 2 copies cell -1 , and the toxin gene copy number showed a proportional relationship not only with the number of blue-green algae cells but also with the concentration of each toxin. However, since the concentration of toxins may vary depending on the growth stage of the target algae or environmental conditions such as nutrients and water temperature (Chiu et al., 2017; Orr et al.. 2010), accurate toxin gene quantitative analysis results are used. Although the toxin concentration cannot be calculated, it is believed that it can be used as data to determine the potential for toxin generation.
독소 유전자 정량분석결과 saxitoxin 합성유전자 보유 남조류 세포수는 최대 50 cells mL-1을 넘지 않았으며, 출현빈도 또한 낮았다. ELISA를 이용한 독소 측정에서도 saxitoxin의 경우 4∼5월 중하류구간에서 미량 검출되었다. 낙동강에서 분리된 strains 중 saxitoxin 생산 strains는 없었다. 하지만, 동일시기에 Aphanizomenon gracile이 일부 출현하였으며, saxitoxin 합성 유전자와 독소검출의 원인 조류로 판단된다(Cires and Ballot, 2016). As a result of quantitative analysis of toxin genes, the maximum number of blue-green algae cells possessing saxitoxin synthesis genes did not exceed 50 cells mL -1 , and the frequency of appearance was also low. In the measurement of toxins using ELISA, trace amounts of saxitoxin were detected in the middle and lower reaches of the river from April to May. Among the strains isolated from the Nakdong River, there were no saxitoxin-producing strains. However, some Aphanizomenon gracile appeared during the same period, and it is believed to be the algae responsible for the saxitoxin synthesis gene and toxin detection (Cires and Ballot, 2016).
독소 특이적 primer를 이용한 유전자 정량분석은 대상 조류의 출현시기, 현존량 및 독소발생 잠재성에 대해 정밀한 분석이 가능한 것으로 나타났다.Quantitative gene analysis using toxin-specific primers was shown to enable precise analysis of the appearance period, current abundance, and toxin generation potential of target birds.
서열목록 전자파일 첨부Sequence list electronic file attached
Claims (12)
서열번호 1 및 서열번호 2의 프라이머; 및 서열번호 3의 프로브를 사용하여 액적 디지털 중합효소 연쇄반응(droplet digital PCR)으로 증폭하는 단계; 및
증폭 결과를 획득하는 단계를 포함하는 삭시톡신 합성 유전자 검출 방법Preparing an isolated DNA or RNA sample;
Primers of SEQ ID NO: 1 and SEQ ID NO: 2; and amplifying by droplet digital polymerase chain reaction (droplet digital PCR) using the probe of SEQ ID NO: 3; and
Saxitoxin synthesis gene detection method comprising the step of obtaining amplification results
제1항 내지 제5항 중 어느 한 항의 프라이머 세트를 사용하여 중합효소 연쇄반응으로 증폭하는 단계; 및
증폭 결과를 획득하는 단계를 포함하는 수중 독소생산 예측 방법Preparing an isolated DNA or RNA sample;
Amplifying by polymerase chain reaction using the primer set of any one of claims 1 to 5; and
Method for predicting toxin production in water including the step of obtaining amplification results
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Non-Patent Citations (2)
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| Seungjun Lee et al., Food Microbiology, 94, 103655, 2020.10.06.(online).* |
| Yong-Jin Kim et al., Harmful Algae, 100, 101954, 2020.11.28(online).* |
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