JP7428326B2 - Method for recovering nucleic acids in specimens - Google Patents
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Description
特許法第３０条第２項適用 ２０２０年（令和２年）１１月１４日～１６日 ｈｔｔｐｓ：／／ｓｉｔｅｓ．ｇｏｏｇｌｅ．ｃｏｍ／ｖｉｅｗ／ｅｄｎａ－ｐｏｐｅｃｏ－２０２０／にて発表Application of Article 30, Paragraph 2 of the Patent Act November 14th to 16th, 2020 (Reiwa 2) https://sites. google. Announced at com/view/edna-popeco-2020/
特許法第３０条第２項適用 ２０２１年（令和３年）２月２６日 ｈｔｔｐｓ：／／ｗｗｗ．ｎａｒｏ．ｇｏ．ｊｐ／ｅｖｅｎｔ／ｌｉｓｔ／２０２１／０１／１３７４３０．ｈｔｍｌにて発表Application of Article 30, Paragraph 2 of the Patent Act February 26, 2021 https://www. naro. go. jp/event/list/2021/01/137430. Announcement in html
本発明は、環境水や土壌中に含まれるＤＮＡを簡易な装置でろ過分離および抽出する技術に関する。 The present invention relates to a technology for filtering, separating and extracting DNA contained in environmental water or soil using a simple device.
環境ＤＮＡを環境水からろ過、分離、抽出する方法は、一般社団法人環境ＤＮＡ学会発行の環境ＤＮＡ調査・実験マニュアル（非特許文献１）に詳細が記載されている。非特許文献１のマニュアルでは、環境水１Ｌから環境ＤＮＡをろ過、分離する方法として、カートリッジ式のフィルターを用いる方法と、ガラス繊維ろ紙を用いる方法の２種類が定義されている。いずれの方法もリアルタイムＰＣＲ法または次世代シーケンサーによる分析を前提としており、ＤＮｅａｓｙ Ｂｌｏｏｄ ａｎｄ Ｔｉｓｓｕｅ Ｋｉｔ（Ｑｉａｇｅｎ）によりＤＮＡを抽出する。 Methods for filtering, separating, and extracting environmental DNA from environmental water are described in detail in the Environmental DNA Investigation and Experiment Manual (Non-Patent Document 1) published by the Environmental DNA Society. The manual of Non-Patent Document 1 defines two methods for filtering and separating environmental DNA from 1 L of environmental water: a method using a cartridge type filter and a method using glass fiber filter paper. Both methods assume analysis using real-time PCR or a next-generation sequencer, and DNA is extracted using a DNeasy Blood and Tissue Kit (Qiagen).
上記した環境ＤＮＡのろ過、分離法は、ＤＮＡやＲＮＡなどの核酸が、カオトロピックの塩の存在下でガラス、シリカゲル、石英ウール、シリカ、ガラス膜又はポリマーなどに吸着する原理を利用したものであり、この原理は一般的に広く知られている。この原理を利用したＤＮＡを抽出する方法としては、ガラス繊維またはガラス粉末を組み込んだカートリッジ式のフィルターによってＤＮＡを精製する方法（例えば、特許第３０４５９２２号公報）、電気泳動後のアガロースゲルからガラス粉末にＤＮＡを吸着させて回収する方法（例えば、特開昭５９－２２７７４４号公報）、ガラス粉末に生体試料のＤＮＡを吸着させて遠心分離により回収する方法（特開平０３－００７５８２号公報）などが報告されている。また、ＤＮＡをシリカ粒子等の懸濁液に吸着させ、メンブレンフィルター上で回収する方法（特許第２６８０４６２号公報及び特開平１０－１５５４８１号公報）も報告されている。 The environmental DNA filtration and separation method described above utilizes the principle that nucleic acids such as DNA and RNA are adsorbed to glass, silica gel, quartz wool, silica, glass membranes, polymers, etc. in the presence of chaotropic salts. , this principle is generally widely known. Methods for extracting DNA using this principle include a method of purifying DNA with a cartridge-type filter incorporating glass fiber or glass powder (for example, Japanese Patent No. 3045922), and a method of purifying DNA with a cartridge-type filter incorporating glass fiber or glass powder (for example, Japanese Patent No. 3045922), and a method of purifying DNA with a cartridge-type filter incorporating glass fiber or glass powder (for example, Japanese Patent No. 3045922), and a method of extracting glass powder from agarose gel after electrophoresis. There are methods of collecting DNA by adsorbing it to glass powder (for example, Japanese Patent Application Laid-Open No. 59-227744), and methods of adsorbing DNA of a biological sample to glass powder and collecting it by centrifugation (Japanese Patent Application Laid-open No. 03-007582). It has been reported. Furthermore, a method has been reported in which DNA is adsorbed to a suspension of silica particles or the like and recovered on a membrane filter (Japanese Patent No. 2680462 and Japanese Patent Application Laid-Open No. 10-155481).
上記した特許文献１～５に記載されているカオトロピック物質にＤＮＡを吸着させて回収する方法は、微量または少量のＤＮＡ溶液を対象としていることから、有機物を含む浮遊物に汚染された清浄ではない水を１Ｌ程度ろ過する必要がある一般的な環境ＤＮＡ分析には適用できない。 The method of collecting DNA by adsorbing it to a chaotropic substance described in Patent Documents 1 to 5 mentioned above is aimed at trace amounts or small amounts of DNA solution, so it is not a clean solution contaminated with suspended matter containing organic matter. It cannot be applied to general environmental DNA analysis, which requires filtering about 1 liter of water.
環境水を対象とする「環境ＤＮＡ調査・実験マニュアル」（非特許文献１）に記載の方法においても、１Ｌを目詰まりさせることなくろ過することはしばしば困難である。同時に、これら従来の方法はいずれも施設の整った実験室で実施する必要があり、広範囲の環境ＤＮＡ調査の物理的な妨げとなっていた。そこで、環境水を実験施設に持ち帰ることなく、採水現場で多量の環境ＤＮＡのろ過分離を可能とする技術の開発が求められていた。 Even in the method described in the "Environmental DNA Survey/Experiment Manual" (Non-Patent Document 1), which targets environmental water, it is often difficult to filter 1 L without clogging. At the same time, all of these conventional methods need to be carried out in well-equipped laboratories, creating a physical impediment to extensive environmental DNA investigations. Therefore, there was a need to develop a technology that would allow large amounts of environmental DNA to be filtered and separated at the water sampling site without having to take the environmental water back to the experimental facility.
本発明は、上述の課題を解決するためになされたものである。即ち、本発明は、簡易な装置で環境ＤＮＡをろ過分離および抽出する方法を提供することを解決すべき課題とする。 The present invention has been made to solve the above-mentioned problems. That is, an object of the present invention is to provide a method for filtering, separating and extracting environmental DNA using a simple device.
本発明者は上記課題を解決するために鋭意検討した結果、水に懸濁したガラス繊維に検体中の核酸を吸着させ、それらのガラス繊維を微細な編み目構造を有するシートで回収することにより、簡易に検体中の核酸をろ過及び分離できることを見出した。本発明によれば、以下の発明が提供される。 As a result of intensive studies to solve the above problems, the inventors of the present invention found that by adsorbing nucleic acids in a sample to glass fibers suspended in water and collecting the glass fibers with a sheet having a fine mesh structure, We have discovered that nucleic acids in a sample can be easily filtered and separated. According to the present invention, the following inventions are provided.
＜１＞ 水に懸濁したガラス繊維に検体中の核酸を吸着させる工程、及び核酸が吸着したガラス繊維を、微細な編み目構造を有するシートでろ過分離することにより回収する工程を含む、検体中の核酸を回収する方法。
＜２＞ 核酸が吸着したガラス繊維を、微細な編み目構造を有するシートでろ過分離することにより回収する工程を、
（ａ）液体を流入するための開口部および液体を排出するための開口部を有する袋、及び
（ｂ）前記の液体を排出するための開口部に設置された、２つの円筒管に挟まれた微細な編み目構造を有するシート、
を有するサンプリングバッグを使用して実施する、＜１＞に記載の方法。
＜３＞ 複数個のサンプリングバッグを並列させて、複数個の検体を同時にろ過する、＜２＞に記載の方法。
＜４＞ 液体を排出するための開口部に吸引ポンプを連結させすることによって、ろ過時間を短縮させる、＜２＞又は＜３＞に記載の方法。
＜５＞ 核酸が吸着したガラス繊維を、微細な編み目構造を有するシートでろ過分離することにより回収する工程を、
液体を流入するための開口部および液体を排出するための開口部を有する吸引カップと、前記の液体を排出するための開口部から排出される液体を受けるための容器とを含み、前記吸引カップと前記容器との間に、微細な編み目構造を有するシートが設置されているＤＮＡろ過器具を使用して実施する、＜１＞に記載の方法。
＜６＞ 複数個のＤＮＡろ過器具を並列させて、複数個の検体を同時にろ過する、＜５＞に記載の方法。
＜７＞ 液体を排出するための開口部に吸引ポンプを連結させすることによって、ろ過時間を短縮させる、＜５＞又は＜６＞に記載の方法。
＜８＞ 微細な編み目構造を有するシートの目開きが、０．１８ｍｍ～０．２８ｍｍである、＜１＞から＜７＞の何れか一に記載の方法。
＜９＞ 核酸を吸着させるためのガラス繊維が、ジルコニアビーズとともに振動数２５Ｈｚで５秒から１５秒粉砕したものである、＜１＞から＜８＞の何れか一に記載の方法。
＜１０＞ 核酸を吸着させるためのガラス繊維の繊維長の分布が、繊維長５０μｍ以下が１０～６５％、繊維長５１μｍ以上３００μｍ以下が３０～６０％、繊維長３０１μｍ以上が５～４０％である、＜１＞から＜９＞の何れか一に記載の方法。
＜１１＞ ＜１＞から＜１０＞の何れか一に記載の方法により検体中の核酸を回収する工程、及び
回収した核酸を用いて、ＬＡＭＰ法によりＤＮＡの増幅を行う工程、
を含む、環境ＤＮＡの検出方法。
＜１２＞ （ａ）液体を流入するための開口部および液体を排出するための開口部を有する袋、及び
（ｂ）前記の液体を排出するための開口部に設置された、２つの円筒管に挟まれた微細な編み目構造を有するシート、
を有するサンプリングバッグ。
＜１３＞ 液体を流入するための開口部および液体を排出するための開口部を有する吸引カップと、前記の液体を排出するための開口部から排出される液体を受けるための容器とを含み、前記吸引カップと前記容器との間に、微細な編み目構造を有するシートが設置されているＤＮＡろ過器具。
<1> A step of adsorbing the nucleic acid in the sample to glass fibers suspended in water, and a step of recovering the glass fibers to which the nucleic acids have been adsorbed by filtering and separating them through a sheet having a fine mesh structure. A method for recovering nucleic acids.
<2> A step of recovering glass fibers to which nucleic acids have been adsorbed by filtering and separating them through a sheet having a fine mesh structure.
(a) a bag having an opening for the inflow of liquid and an opening for the evacuation of the liquid; and (b) sandwiched between two cylindrical tubes placed in the opening for the evacuation of said liquid. A sheet with a fine mesh structure,
The method according to <1>, which is carried out using a sampling bag having:
<3> The method according to <2>, wherein a plurality of sampling bags are arranged in parallel to filter a plurality of samples at the same time.
<4> The method according to <2> or <3>, wherein the filtration time is shortened by connecting a suction pump to the opening for discharging the liquid.
<5> A step of recovering glass fibers to which nucleic acids have been adsorbed by filtering and separating them through a sheet having a fine mesh structure,
a suction cup having an opening for entering liquid and an opening for discharging liquid; and a container for receiving liquid discharged from the opening for discharging said liquid; The method according to <1>, which is carried out using a DNA filtration device in which a sheet having a fine mesh structure is installed between the DNA filtration device and the container.
<6> The method according to <5>, wherein a plurality of DNA filtration devices are arranged in parallel to filter a plurality of samples at the same time.
<7> The method according to <5> or <6>, wherein the filtration time is shortened by connecting a suction pump to the opening for discharging the liquid.
<8> The method according to any one of <1> to <7>, wherein the sheet having a fine mesh structure has an opening of 0.18 mm to 0.28 mm.
<9> The method according to any one of <1> to <8>, wherein the glass fibers for adsorbing nucleic acids are pulverized together with zirconia beads at a frequency of 25 Hz for 5 to 15 seconds.
<10> The fiber length distribution of the glass fiber for adsorbing nucleic acids is such that 10 to 65% have a fiber length of 50 μm or less, 30 to 60% have a fiber length of 51 μm or more and 300 μm or less, and 5 to 40% have a fiber length of 301 μm or more. The method according to any one of <1> to <9>.
<11> A step of recovering nucleic acid in a specimen by the method described in any one of <1> to <10>, and a step of amplifying DNA by the LAMP method using the recovered nucleic acid,
A method for detecting environmental DNA.
<12> (a) A bag having an opening for inflowing a liquid and an opening for discharging a liquid, and (b) two cylindrical tubes installed in the opening for discharging the liquid. A sheet with a fine mesh structure sandwiched between
A sampling bag with.
<13> A suction cup having an opening for inflowing a liquid and an opening for discharging the liquid, and a container for receiving the liquid discharged from the opening for discharging the liquid, A DNA filtration device, wherein a sheet having a fine mesh structure is installed between the suction cup and the container.
本発明によれば、土壌懸濁水を含む環境水中のＤＮＡを目詰まりすることなく容易にろ過分離できる。このろ過物は市販の簡易な試薬でＤＮＡ抽出でき、こうして抽出されたＤＮＡはＰＣＲ法以外にもＬＡＭＰ法で分析可能である。これにより、環境ＤＮＡ分析をオンサイトで実施でき、分析効率が飛躍的に向上する。 According to the present invention, DNA in environmental water including soil suspension water can be easily filtered and separated without clogging. DNA can be extracted from this filtrate using a simple commercially available reagent, and the DNA thus extracted can be analyzed by the LAMP method in addition to the PCR method. This allows environmental DNA analysis to be carried out on-site, dramatically improving analysis efficiency.
以下、本発明の実施の形態について、説明する。
本発明による検体中の核酸を回収する方法は、水に懸濁したガラス繊維に検体中の核酸を吸着させる工程、及び核酸が吸着したガラス繊維を、微細な編み目構造を有するシートでろ過分離することにより回収する工程を含む方法である。
Embodiments of the present invention will be described below.
The method of recovering nucleic acids in a specimen according to the present invention includes a step of adsorbing nucleic acids in a specimen to glass fibers suspended in water, and filtering and separating the glass fibers to which nucleic acids have been adsorbed using a sheet having a fine mesh structure. This method includes a step of recovering the waste.
本発明においては、検体としては環境から採取した検体を使用することができる。環境とは、土壌、堆肥、汚泥、下水、河川、湖沼、海水、海底、昆虫、動物、及び植物の固体の組織内部及び表面、並びに、排泄物等、微生物の生育環境を意味する。環境から採取した検体とは、具体的には、土壌、堆肥、汚泥、下水、河川、湖沼、海水、海底、昆虫、動物、及び植物の固体の組織内部及び表面、並びに、排泄物等から採取された検体が挙げられる。 In the present invention, a specimen collected from the environment can be used as the specimen. The environment refers to the environment in which microorganisms grow, such as soil, compost, sludge, sewage, rivers, lakes, seawater, the seabed, the inside and surface of solid tissues of insects, animals, and plants, and excreta. Specimens collected from the environment include, specifically, samples collected from soil, compost, sludge, sewage, rivers, lakes, seawater, the seabed, insects, animals, the inside and surface of solid tissue of plants, and excreta. Examples include specimens that have been tested.
具体的には、市販のガラス繊維ろ紙を少量の水中で破砕して懸濁し、それを検体に加えることで、検体中のＤＮＡをガラス繊維に吸着させる。その後、樹脂製または天然素材のガーゼ等の微細な編み目構造を有するシートをフィルターとして、ガラス繊維をろ過、回収する。 Specifically, by crushing and suspending commercially available glass fiber filter paper in a small amount of water and adding it to the sample, the DNA in the sample is adsorbed onto the glass fibers. Thereafter, the glass fibers are filtered and recovered using a sheet having a fine mesh structure, such as gauze made of resin or natural material, as a filter.
微細な編み目構造を有するシートは、懸濁したガラス繊維を補足するだけの目開きがあればよく、先行技術でみられるようなメンブレンフィルターやガラス繊維ろ紙のような極微細な目開きである必要はない。逆に目開きが微細すぎるとろ過時に目詰まりを生じがちであるし、また、広すぎるとガラス繊維が捕捉されずに流出してしまい、回収できない。従って、適度な目開きのフィルターを用いることで池や川などの清浄でない水をろ過する場合でも、目詰まりすることはなくガラス繊維を捕捉、回収できる。 The sheet with a fine mesh structure only needs to have an opening large enough to capture the suspended glass fibers, and it is not necessary that the sheet has an extremely fine opening such as membrane filters and glass fiber filter papers seen in the prior art. There isn't. On the other hand, if the openings are too fine, clogging tends to occur during filtration, and if the openings are too wide, the glass fibers will flow out without being captured, making it impossible to recover them. Therefore, by using a filter with an appropriate opening, even when filtering unclean water from ponds, rivers, etc., glass fibers can be captured and recovered without clogging.
微細な編み目構造を有するシートの目開きは、一般的には、０．０７ｍｍ～０．５０ｍｍあり、好ましくは０．１０ｍｍ～０．４０ｍｍであり、好ましくは０．１８ｍｍ～０．２８ｍｍである。 The opening of the sheet having a fine mesh structure is generally 0.07 mm to 0.50 mm, preferably 0.10 mm to 0.40 mm, and preferably 0.18 mm to 0.28 mm.
核酸を吸着させるためのガラス繊維としては、ジルコニアビーズとともに振動数２５Ｈｚで５秒から１５秒粉砕したものを使用することが好ましい。 As the glass fiber for adsorbing nucleic acids, it is preferable to use glass fibers that have been crushed together with zirconia beads at a frequency of 25 Hz for 5 to 15 seconds.
核酸を吸着させるためのガラス繊維の繊維長の分布としては、好ましくは、繊維長５０μｍ以下が１０～６５％（より好ましくは１０～３０％）、繊維長５１μｍ以上３００μｍ以下が３０～６０％（より好ましくは５０～６０％）、繊維長３０１μｍ以上が５～４０％（より好ましくは１０～３０％）であることが好ましい。 The fiber length distribution of the glass fiber for adsorbing nucleic acids is preferably 10-65% (more preferably 10-30%) with a fiber length of 50 μm or less, and 30-60% (more preferably 10-30%) with a fiber length of 51 μm or more and 300 μm or less. (more preferably 50 to 60%), and 5 to 40% (more preferably 10 to 30%) of fibers having a length of 301 μm or more.
本発明では、河川水、池水、海水、など環境中の水に溶存するＤＮＡを現場で採取およびろ過する器具として、一例としては、以下の構成を採用することができる。検体を流入または排出するための開口部を備えた、およそ容量２リットル以下を目安としたポリエチレン製またはその他の合成樹脂製のサンプリングバックを用い、開口部に塩化ビニル製またはその他の合成樹脂製からなる円筒管を１個または複数個連結し、間にメッシュシートまたはガーゼまたはその他微細な編み目構造を有するシートを装着する。これを簡易ＤＮＡろ過用のサンプリングバッグとする。 In the present invention, as an example, the following configuration can be adopted as a device for collecting and filtering DNA dissolved in environmental water such as river water, pond water, seawater, etc. on site. Use a sampling bag made of polyethylene or other synthetic resin with a capacity of approximately 2 liters or less, equipped with an opening for inflowing or discharging the sample, and with an opening made of vinyl chloride or other synthetic resin. One or more cylindrical tubes are connected, and a mesh sheet, gauze, or other sheet having a fine mesh structure is attached between them. This is used as a sampling bag for simple DNA filtration.
また、土壌懸濁水などの対象の検体が少量の場合は、上記のサンプリングバッグ以外にも市販のろ過瓶にメッシュシート等を介して重力ろ過することもできる。 In addition, when the target sample such as soil suspension water is small, gravity filtration can be performed using a commercially available filter bottle through a mesh sheet or the like instead of the above-mentioned sampling bag.
環境水を入れたサンプリングバックまたは土壌懸濁水などにあっては小容量の容器に、懸濁または溶解したガラス繊維を適量加えて混合する。添加するガラス繊維は、メーカー、規格は問わず、また、容器に少量の水と規定量のガラス繊維ろ紙を加えて、破砕用ビーズとともに激しく振ることで、繊維が切断されず懸濁させることができる。ガラス繊維を混合することで検体中のＤＮＡがガラス繊維に吸着保持されるため、サンプリングバッグを開口部が下部になるように保持することで、ガラス繊維以外の有機物を含む浮遊物がメッシュシートの網目を通過して排水され、ＤＮＡを吸着したガラス繊維を含むろ過物がメッシュシート上に堆積する。このとき、サンプリングバッグの側面に圧力を加えることで、ろ過時間を短縮することができる。 For sampling bags containing environmental water or soil suspension water, add an appropriate amount of suspended or dissolved glass fibers to a small container and mix. The glass fibers to be added can be of any manufacturer or standard, and by adding a small amount of water and a specified amount of glass fiber filter paper to a container and shaking them vigorously together with crushing beads, the fibers can be suspended without being cut. can. By mixing glass fibers, the DNA in the sample is adsorbed and retained by the glass fibers, so by holding the sampling bag with the opening at the bottom, floating matter containing organic matter other than glass fibers can be absorbed by the mesh sheet. Drainage passes through the mesh, and a filtrate containing glass fibers adsorbing DNA is deposited on the mesh sheet. At this time, the filtration time can be shortened by applying pressure to the side of the sampling bag.
この方法は、サンプリングバッグまたはフィルターをフック等による吊り下げまたは架台等に一定間隔で列状に並べることで、複数個の検体を同時にろ過することも可能である。 In this method, it is also possible to filter a plurality of samples at the same time by hanging sampling bags or filters from hooks or the like or by arranging them in rows at regular intervals on a stand or the like.
また、サンプリングバッグの排水側にポンプを接続して吸引ろ過を行い、ろ過時間を短縮させることもできる。 It is also possible to connect a pump to the drainage side of the sampling bag and perform suction filtration to shorten the filtration time.
ろ過物は、例えばＭｉｇｈｔｙＰｒｅｐ ｒｅａｇｅｎｔ ｆｏｒ ＤＮＡ （Ｔａｋａｒａ）等の簡易ＤＮＡ抽出試薬でＤＮＡ抽出が可能である。 DNA can be extracted from the filtrate using a simple DNA extraction reagent such as MightyPrep reagent for DNA (Takara).
上記で抽出されたＤＮＡは、核酸増幅反応に供することができる。
核酸増幅反応としては、当業者にとって公知の方法を用いることができ、例えば、ポリメラーゼ連鎖反応（ＰＣＲ法）が挙げられる。ＰＣＲ法においては、環境ＤＮＡを鋳型として、特定のプライマーセットを用いてＰＣＲを行うことにより増幅産物を得ることができる。ＰＣＲ法は、リアルタイムＰＣＲ法でもよい。
The DNA extracted above can be subjected to a nucleic acid amplification reaction.
As the nucleic acid amplification reaction, methods known to those skilled in the art can be used, such as polymerase chain reaction (PCR method). In the PCR method, an amplification product can be obtained by performing PCR using environmental DNA as a template and a specific primer set. The PCR method may be a real-time PCR method.
また、核酸増幅反応としては、ＰＣＲ法だけではなく、特殊な分析装置を必要としない核酸増幅法であるｌｏｏｐ－ｍｅｄｉａｔｅｄ ｉｓｏｔｈｅｒｍａｌ ａｍｐｌｉｆｉｃａｔｉｏｎ（ＬＡＭＰ）法を行ってもよい。ＬＡＭＰ法は、標的遺伝子の配列から６つ～８つの領域を選んで組み合わせた４種類～６種類のプライマーを用いて、鎖置換反応を利用して増幅させる方法である。プライマーの設計によって、最初の増幅産物のプライマー結合部位にループ構造を生じるようになる。ループ部分は１本鎖なので、次のプライマーが結合できる。鎖置換活性の高い特殊なＤＮＡ合成酵素は、進行方向にある２本鎖ＤＮＡを解離しながら、自らの伸長反応を進めていく。最終的には、もとの標的配列の約整数倍の長さの増幅産物が１時間ほどの約６５℃の反応で蓄積する。したがって反応産物を電気泳動するとラダー状である。ＰＣＲ法と比較して、１本鎖から２本鎖への変性反応が必要なく、６０～７０℃の定温または６５℃前後の定温で反応が進行することから、サーマルサイクラーは不要である。また、増幅速度が速く、特異性も高いことから、反応液の白濁を見るだけでテンプレート（標的ＤＮＡ）が増えたかどうかを確認できる。 Furthermore, as the nucleic acid amplification reaction, not only the PCR method but also the loop-mediated isothermal amplification (LAMP) method, which is a nucleic acid amplification method that does not require a special analysis device, may be performed. The LAMP method is a method of amplification using a strand displacement reaction using four to six types of primers selected and combined from six to eight regions from the sequence of a target gene. The design of the primers creates a loop structure at the primer binding site of the initial amplification product. Since the loop portion is single-stranded, the next primer can bind to it. A special DNA synthesizing enzyme with high strand displacement activity proceeds with its own elongation reaction while dissociating double-stranded DNA in the direction of propagation. Ultimately, amplification products approximately integer times the length of the original target sequence accumulate in a reaction time of about 65° C. for about 1 hour. Therefore, when the reaction products are electrophoresed, they appear in a ladder shape. Compared to the PCR method, there is no need for a denaturation reaction from single strands to double strands, and the reaction proceeds at a constant temperature of 60 to 70°C or around 65°C, so a thermal cycler is not necessary. Furthermore, since the amplification rate is fast and the specificity is high, it is possible to confirm whether the template (target DNA) has increased simply by observing the cloudiness of the reaction solution.
以下に、ガラス繊維を用いたＤＮＡの採取・ろ過法の概要、フィールド等における環境水の採取・ろ過法、および土壌懸濁水のろ過法に分けて、図面に基づいて説明する。 Below, an outline of a method for collecting and filtering DNA using glass fibers, a method for collecting and filtering environmental water in the field, and a method for filtering soil suspended water will be explained based on drawings.
ガラス繊維を用いたＤＮＡの採取・ろ過法においては、図１に示す破砕して水に懸濁したガラス繊維を環境水１Ｌに加えて撹拌し、従来の水のろ過用フィルターホルダーなどにメッシュシートを固定し、ガラス繊維を混合した環境水を通水させてろ過し、メッシュシート上に堆積したガラス繊維（図２）を回収する。 In the DNA collection and filtration method using glass fibers, the glass fibers shown in Figure 1, which have been crushed and suspended in water, are added to 1 L of environmental water, stirred, and placed in a mesh sheet in a conventional water filtration filter holder. is fixed, and environmental water mixed with glass fibers is passed through and filtered to collect the glass fibers deposited on the mesh sheet (Figure 2).
ガラス繊維の規格やメーカーは問わない。ガラス繊維の量は、実験の結果、５ｍｇ～２０ｍｇが望ましいことが明らかとなっている。また、ガラス繊維は破砕方法によっては繊維が短くなりすぎ、メッシュシートを通過して回収できない場合があるため、繊維が分断されすぎない程度に破砕し、さらにそれらを回収するのに適した目開きのメッシュシートを組み合わせる必要がある。 The standard and manufacturer of glass fiber does not matter. Experiments have shown that the amount of glass fiber is preferably 5 mg to 20 mg. In addition, depending on the crushing method, the glass fibers may become too short and cannot be collected by passing through the mesh sheet, so it is necessary to crush the fibers to the extent that the fibers are not too divided, and also have a mesh opening suitable for collecting them. It is necessary to combine the mesh sheets.
図３は、（ａ）液体を流入するための開口部および液体を排出するための開口部を有する袋、及び（ｂ）前記の液体を排出するための開口部に設置された、２つの円筒管に挟まれた微細な編み目構造を有するシート、を有するサンプリングバッグの一例を示す。図３の装置は、キャップ付きサンプリングバッグ１、ホース２、シリコン栓３、キャップに装着する塩化ビニル製のアダプタ４ 、メッシュシート５ 、メッシュシート固定用の塩化ビニル製のアダプタ６から構成されている。図３のサンプリングバッグは、フィールド等における環境水の採取・ろ過法において使用することができる。 FIG. 3 shows (a) a bag having an opening for inlet of liquid and an opening for discharging liquid; and (b) two cylinders installed in said opening for discharging liquid. An example of a sampling bag having a sheet having a fine mesh structure sandwiched between tubes is shown. The apparatus shown in Fig. 3 is composed of a sampling bag 1 with a cap, a hose 2, a silicone stopper 3, a vinyl chloride adapter 4 attached to the cap, a mesh sheet 5, and a vinyl chloride adapter 6 for fixing the mesh sheet. . The sampling bag shown in FIG. 3 can be used in environmental water collection and filtration methods in the field and the like.
キャップ付きサンプリングバッグ１は環境水と懸濁させたガラス繊維を混合するための容器である。採取した環境水１Ｌをサンプリングバック１に入れ、懸濁させたガラス繊維を加え、良く撹拌する。その後、ろ過用のアタッチメントを装着する。ろ過用のアタッチメントは次の通りである。すなわり、サンプリングバッグの開口部内径と一致する外径の水道用ホース２をシリコン栓３を介して、アダプタ４に接続する。メッシュシート５を被せ、さらにその上からアダプタ６でメッシュシートを固定する。装着後、サンプリングバッグとろ過用のアタッチメントを反転させ、環境水を重力ろ過する。環境水の濁りが酷い場合は、重力ろ過だけではろ過できないことが多いため、手でバックの側面を押さえて加圧しながら環境水を排出する。サンプリングバッグ内の環境水のおおよそを排出した後、手でバッグを押さえて、バッグおよびろ過用のアタッチメント内の環境水を完全に排出する。その後、アダプタ６をゆっくり外して、メッシュシートを取り出す。メッシュシート上には図３のように、ＤＮＡを含んだガラス繊維がフィルム状に堆積しており、それを滅菌したつまようじまたはピンセットなどで慎重に巻き取り、０．２ｍＬ～１．５ｍＬのＰＣＲチューブ等に入れる。ここまでがフィールド等における環境水の採取・ろ過法である。 The sampling bag 1 with a cap is a container for mixing environmental water and suspended glass fibers. Put 1L of the collected environmental water into sampling bag 1, add the suspended glass fibers, and stir well. Then, attach the filtration attachment. The attachments for filtration are as follows. That is, a water hose 2 having an outer diameter matching the inner diameter of the opening of the sampling bag is connected to the adapter 4 via the silicon plug 3. A mesh sheet 5 is placed over it, and the mesh sheet is further fixed with an adapter 6 from above. After installation, the sampling bag and filtration attachment are inverted and environmental water is gravity filtered. If the environmental water is extremely cloudy, gravity filtration alone may not be enough to filter it, so press the side of the bag with your hand and pressurize it while draining the environmental water. After approximately draining the environmental water in the sampling bag, hold the bag with your hand to completely drain the environmental water in the bag and the filtration attachment. After that, slowly remove the adapter 6 and take out the mesh sheet. As shown in Figure 3, glass fibers containing DNA are deposited on the mesh sheet in the form of a film, which is carefully rolled up with a sterilized toothpick or tweezers and placed in a 0.2 mL to 1.5 mL PCR tube. etc. This is the method for collecting and filtering environmental water in the field.
図４は、液体を流入するための開口部および液体を排出するための開口部を有する吸引カップと、前記の液体を排出するための開口部から排出される液体を受けるための容器とを含み、前記吸引カップと前記容器との間に、微細な編み目構造を有するシートが設置されているＤＮＡろ過器具の一例を示す。図４のＤＮＡろ過器具は、吸引カップ１１、メッシュシート１２、吸引瓶１３から構成されている。図４のＤＮＡろ過器具は、土壌懸濁水のろ過法において使用することができる。 FIG. 4 includes a suction cup having an opening for admitting liquid and an opening for discharging liquid, and a container for receiving liquid discharged from said opening for discharging liquid. , shows an example of a DNA filtration device in which a sheet having a fine mesh structure is installed between the suction cup and the container. The DNA filtration device shown in FIG. 4 is composed of a suction cup 11, a mesh sheet 12, and a suction bottle 13. The DNA filtration device of FIG. 4 can be used in a soil suspension water filtration method.
土壌を加えて撹拌した水にガラス繊維を適量加えてよく撹拌する。実験を重ねた結果、約１００ｍＬの水に土壌を０．５ｇ加えて良く撹拌し、約５ｍｇのガラス繊維を加えるとよいことが明らかとなっている。メッシュシートを挟んだ吸引カップを吸引瓶にセットし、ガラス繊維を加えた土壌懸濁水を注ぎ入れる。このとき、重力のみでろ過できるので、必ずしもポンプ等で吸引する必要はない。ろ過後に吸引カップを外してメッシュシートを回収する。その後は上述の方法と同様にしてフィルム状に堆積したＤＮＡを含むガラス繊維をＰＣＲチューブ等に入れる。 Add an appropriate amount of glass fiber to the water that has been stirred with soil and stir well. As a result of repeated experiments, it has become clear that it is best to add 0.5 g of soil to about 100 mL of water, stir well, and add about 5 mg of glass fiber. A suction cup with a mesh sheet sandwiched in between is set in a suction bottle, and soil suspension water containing glass fiber is poured into it. At this time, since filtration can be performed only by gravity, it is not necessarily necessary to suction with a pump or the like. After filtration, remove the suction cup and collect the mesh sheet. Thereafter, glass fibers containing DNA deposited in the form of a film are placed in a PCR tube or the like in the same manner as described above.
以下の実施例により本発明をより詳細に説明するが、本発明の範囲はこれらの実施例に限定されるものではない。 The present invention will be explained in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.
＜１＞ガラス繊維の性状とそれを回収するためのメッシュシートの組み合わせ
添加するガラス繊維を効率良く回収できるメッシュシートの目開きを明らかにするために、はじめに、破砕方法の違いによるガラス繊維の性状（長さ）を計測した。ガラス繊維は（ＧＡ－５５、Ａｄｖａｎｔｅｃ）を用い、ガラス繊維１０ｍｇに１ｍｌのＤＷを加え、ＴｉｓｓｕｅＬｙｓｅｒ（Ｑｉａｇｅｎ）を用いて、約５ｍｍのジルコニアボールとともに振動数２５Ｈｚで５秒、１５秒、６０秒破砕した場合およびビニル袋内で手もみした場合の４通りの破砕を行い、その繊維長を光学顕微鏡で計測した。
<1> Properties of glass fibers and combinations of mesh sheets for recovering them In order to clarify the opening of mesh sheets that can efficiently recover added glass fibers, we first examine the properties of glass fibers depending on the different crushing methods. (length) was measured. For glass fibers, 1ml of DW was added to 10mg of glass fibers using (GA-55, Advantec), and crushed using a TissueLyser (Qiagen) with a zirconia ball of about 5mm at a frequency of 25Hz for 5 seconds, 15 seconds, and 60 seconds. The fibers were crushed in four ways: when the fibers were rubbed by hand in a plastic bag, and when they were kneaded by hand in a plastic bag, and the fiber lengths were measured using an optical microscope.
次に、目開きが１．０ｍｍ、０．２８ｍｍ、０．１８ｍｍ、０．０７ｍｍの４種類のメッシュシートを用いて、上記の破砕方法で調製したガラス繊維１０ｍｇを懸濁した水道水をろ過し、フィルター上に残ったガラス繊維の重量（ガラス繊維捕捉率）を測定した。実験は３回実施した。 Next, tap water in which 10 mg of glass fiber prepared by the above crushing method was suspended was filtered using four types of mesh sheets with openings of 1.0 mm, 0.28 mm, 0.18 mm, and 0.07 mm. The weight of the glass fibers remaining on the filter (glass fiber capture rate) was measured. The experiment was performed three times.
次に、目詰まりしないメッシュシートを選定するために、浮遊物の多い池の水１Ｌを上記の破砕方法で調製したガラス繊維およびメッシュシートの様々な組み合わせでろ過を行い、目詰まりの有無およびろ過時間を計測した。このときのろ過時間は５秒以内に連続して滴下が確認できなくなるまでの時間と定義した。ただし、目開き１．０ｍｍのフィルターはこれまでの実験から使用に適さないことが明らかだったため、対象から除外した。実験は３回実施した。 Next, in order to select a mesh sheet that does not clog, 1 liter of pond water with a lot of suspended matter was filtered through various combinations of glass fibers and mesh sheets prepared using the above crushing method, and the presence or absence of clogging was determined. I measured the time. The filtration time at this time was defined as the time until continuous dripping could no longer be confirmed within 5 seconds. However, it was clear from previous experiments that the filter with an opening of 1.0 mm was not suitable for use, so it was excluded from the test. The experiment was performed three times.
＜２＞環境ＤＮＡの収量の比較（１）：ガラス繊維の調製法とメッシュシートの決定
５秒、１５秒、６０秒破砕したガラス繊維を使って、カワヒバリガイ飼育水に含まれるカワヒバリガイ環境ＤＮＡをろ過、分離し、ＤＮＡ抽出量を比較した。カワヒバリガイ飼育水は、カワヒバリガイ約５０匹を飼育している水量約６０Ｌの水を用いた。飼育水１Ｌに破砕懸濁したガラス繊維ＧＡ－５５を１０ｍｇ加え、良く撹拌した後に、０．２８ｍｍ、０．１８ｍｍ、０．０７ｍｍの３種類のメッシュシートでろ過し、ガラス繊維を回収した。ＭｉｇｈｔｙＰｒｅｐ Ｒｅａｇｅｎｔ ｆｏｒ ＤＮＡ（Ｔａｋａｒａ）でＤＮＡを抽出し、リアルタイムＰＣＲ装置ＣＦＸ９６（ＢｉｏＲａｄ）を用いて、カワヒバリガイ特異的ＰＣＲプライマー（Ｘｉａ ｅｔ ａｌ， ２０１８）によりＰＣＲを行い、カワヒバリガイ環境ＤＮＡを定量した。実験は３回実施した。
<2> Comparison of yield of environmental DNA (1): Determination of glass fiber preparation method and mesh sheet Using glass fibers crushed for 5 seconds, 15 seconds, and 60 seconds, filter the environmental DNA of Kawahibari mussels contained in Kawahibari mussel rearing water , and the amount of DNA extracted was compared. The water used for rearing about 60 liters of water for breeding about 50 mussels was used. 10 mg of crushed and suspended glass fiber GA-55 was added to 1 L of rearing water, stirred well, and then filtered through three types of mesh sheets of 0.28 mm, 0.18 mm, and 0.07 mm to collect glass fibers. DNA was extracted with MightyPrep Reagent for DNA (Takara), and PCR was performed using a real-time PCR device CFX96 (BioRad) with a C. laurifolia specific PCR primer (Xia et al, 2018) to quantify C. laurifolia environmental DNA. The experiment was performed three times.
Xia Z, Zhan A, Gao Y, Zhang L, Haffner DG, MacIsaac HJ (2018) Early detection of a highly invasive bivalve based on environmental DNA (eDNA). Biol Invasions 20: 437-447. doi:org/10.1007/s10530-017-1545-7 Xia Z, Zhan A, Gao Y, Zhang L, Haffner DG, MacIsaac HJ (2018) Early detection of a highly invasive bivalve based on environmental DNA (eDNA). Biol Invasions 20: 437-447. doi:org/10.1007/s10530 -017-1545-7
＜３＞環境ＤＮＡの収量の比較（２）：ガラス繊維の添加量の決定
ガラス繊維により環境水中のＤＮＡが吸着される効果を明らかにするために、カワヒバリガイ飼育水を用いて実験を行った。カワヒバリガイ飼育水は、カワヒバリガイ約２０匹を飼育している水量約６０Ｌの水を用いた。飼育水１Ｌに破砕時間１５秒で破砕懸濁させたガラス繊維ＧＡ－５５を５ｍｇ～５０ｍｇ加え、良く撹拌した後に、１００メッシュのステンレススクリーンを通過させて、ガラス繊維を取り除いた。その後、環境ＤＮＡ調査・実験マニュアル（一般社団法人環境ＤＮＡ学会）に基づいて飼育水中のカワヒバリガイ環境ＤＮＡをろ過・抽出した。すなわち、孔径０．７μｍのガラス繊維ＧＦ／Ｆ（Ｗｈａｔｍａｎ）を用いて吸引ろ過を行い、回収したＧＦ／ＦからＤＮｅａｓｙ Ｂｌｏｏｄ ａｎｄ Ｔｉｓｓｕｅ ｋｉｔによりＤＮＡを抽出した。リアルタイムＰＣＲ装置ＣＦＸ９６を用いて、カワヒバリガイ特異的ＰＣＲプライマー（Ｘｉａ ｅｔ ａｌ， ２０１８）によりＰＣＲを行い、カワヒバリガイ環境ＤＮＡを定量した。吸着ＤＮＡはガラス繊維を添加しなかった飼育水の抽出ＤＮＡから、ガラス繊維を添加した後に除去した飼育水の抽出ＤＭＡを差し引いて求め、吸着率を算出した。実験は４回実施した。
<3> Comparison of the yield of environmental DNA (2): Determination of the amount of glass fiber added In order to clarify the effect of adsorption of DNA in environmental water by glass fiber, an experiment was conducted using water for breeding snails. The water used for rearing about 60 liters of water for breeding about 20 mussels was used. 5 mg to 50 mg of crushed and suspended glass fiber GA-55 was added to 1 L of rearing water for a crushing time of 15 seconds, and after stirring well, the mixture was passed through a 100 mesh stainless steel screen to remove the glass fibers. Thereafter, the environmental DNA of the Japanese snail in the rearing water was filtered and extracted based on the Environmental DNA Research and Experiment Manual (General Incorporated Association, Environmental DNA Society). That is, suction filtration was performed using glass fiber GF/F (Whatman) with a pore size of 0.7 μm, and DNA was extracted from the collected GF/F using a DNeasy Blood and Tissue kit. Using a real-time PCR device CFX96, PCR was performed using C. laurifolia specific PCR primers (Xia et al, 2018), and C. laurifolia environmental DNA was quantified. The adsorbed DNA was determined by subtracting the extracted DMA of the rearing water that was removed after adding glass fibers from the DNA extracted from the rearing water to which no glass fibers had been added, and the adsorption rate was calculated. The experiment was performed four times.
＜４＞各種ガラス繊維によるＰＣＲ法およびＬＡＭＰ法による環境ＤＮＡ検出
Ｗｈａｔｍａｎ社、ＡＤＶＡＮＴＥＣ社、アズワンの３つのメーカーから複数の規格のガラス繊維ろ紙を選び、それぞれ１０ｍｇを破砕時間１５秒で破砕懸濁してカワヒバリガイ飼育水１Ｌに添加した。図３に示す本発明の簡易ろ過法（メッシュシート 目開き０．１８ｍｍ）でガラス繊維を回収し、ＭｉｇｈｔｙＰｒｅｐ Ｒｅａｇｅｎｔ ｆｏｒ ＤＮＡでＤＮＡを抽出した。リアルタイムＰＣＲによりＣｔ値を測定するとともにカワヒバリガイ特異的なＬＡＭＰプライマーを用いてＬＡＭＰ反応を行った。カワヒバリガイ特異的ＬＡＭＰプライマーとして、ＧｅｎＢａｎｋから得たカワヒバリガイのｍｔＣＯＩ遺伝子配列（Ａｃｃｅｓｓｉｏｎ Ｎｏ．ＡＢ５２０６２７，ＤＱ２６４３９５，ＪＸ１７７０８７）を基に、ＰｒｉｍｅｒＥｘｐｌｏｒｅｒ Ｖｅｒ．５（ 栄研化学 http://primerexplorer.jp/）を用いて、Ｆ３、Ｂ３、ＦＩＰ、ＢＩＰおよびＦｌｏｏｐから構成されるプライマーセットを設計した（表１）。ＬＡＭＰ反応液は、２０ｍＭ Ｔｒｉｓ－ＨＣｌ（ｐＨ８．８）、１０ｍＭ ＫＣｌ、１０ｍＭ （ＮＨ４）２ＳＯ４、０．１％ Ｔｗｅｅｎ２０、０．８Ｍ ｂｅｔａｉｎ（ＦＵＪＩＦＩＬＭ Ｗａｋｏ）、８ｍＭ ＭｇＳＯ４、１．４ｍＭ ｄＮＴＰｓ、０．２μＭ Ｆ３およびＢ３ プライマー、１．６ μＭ ＦＩＰ およびＢＩＰプライマー、０．８μＭ Ｆｌｏｏｐプライマー、８ｕｎｉｔｓ Ｂｓｔ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（Ｎｉｐｐｏｎ Ｇｅｎｅ）を混合し、滅菌水およびＤＮＡ抽出液１ μｌを加えて、総量２０μｌとした。ＬＡＭＰ反応は、リアルタイム濁度計ＬＡ２００（Ｔｅｒａｍｅｃｓ）を用い、濁度が閾値０．１に達した時間を計測した。
<4> Detection of environmental DNA by PCR method and LAMP method using various glass fibers Glass fiber filter papers of multiple standards were selected from three manufacturers: Whatman, ADVANTEC, and As One, and 10 mg of each was crushed and suspended in a crushing time of 15 seconds. It was added to 1 liter of water for breeding the Japanese snail. Glass fibers were collected using the simple filtration method of the present invention (mesh sheet opening 0.18 mm) shown in FIG. 3, and DNA was extracted using MightyPrep Reagent for DNA. The Ct value was measured by real-time PCR, and a LAMP reaction was performed using LAMP primers specific to the snail. Primer Explorer Ver. 5 (Eiken Chemical http://primerexplorer.jp/), a primer set consisting of F3, B3, FIP, BIP, and Floop was designed (Table 1). The LAMP reaction solution contained 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 0.1% Tween 20, 0.8M betain (FUJIFILM Wako), 8mM MgSO 4 , 1.4mM dNTPs. , 0.2 μM F3 and B3 primers, 1.6 μM FIP and BIP primers, 0.8 μM Floop primer, 8 units Bst DNA polymerase (Nippon Gene), and add sterile water and 1 μl of DNA extract solution to make a total volume of 20 μl. And so. For the LAMP reaction, the time when the turbidity reached the threshold value of 0.1 was measured using a real-time turbidimeter LA200 (Teramecs).
＜５＞カワヒバリガイ環境ＤＮＡのＰＣＲ検出およびＬＡＭＰ検出の比較（室内実験）
カワヒバリガイ飼育水１Ｌを簡易ろ過法（ガラス繊維ろ紙ＧＡ－５５、添加量１０ｍｇ、１５秒破砕、０．１８ｍｍメッシュシート）と従来法（環境ＤＮＡ調査・実験マニュアル）でろ過し、前者についてはＭｉｇｈｔｙＰｒｅｐ Ｒｅａｇｅｎｔ ｆｏｒ ＤＮＡ で、後者についてはＤＮｅａｓｙ Ｂｌｏｏｄ ａｎｄ Ｔｉｓｓｕｅ ｋｉｔによりＤＮＡを抽出した。リアルタイムＰＣＲによりＣｔ値を測定するとともにカワヒバリガイ特異的なＬＡＭＰプライマーを用いてＬＡＭＰ反応を行い、リアルタイム濁度計ＬＡ２００を用いて反応時間を計測し、３０分以内に濁度が閾値０．１に達した場合を陽性とした。試験は２０回実施した。
<5> Comparison of PCR detection and LAMP detection of Kawahibari mussel environmental DNA (indoor experiment)
Filter 1L of water for breeding snails using a simple filtration method (Glass fiber filter paper GA-55, added amount 10mg, crushed for 15 seconds, 0.18mm mesh sheet) and a conventional method (Environmental DNA investigation/experiment manual).For the former, use MightyPrep Reagent. For the latter, DNA was extracted using the DNeasy Blood and Tissue kit. The Ct value was measured by real-time PCR, and a LAMP reaction was performed using a LAMP primer specific to the snail, and the reaction time was measured using a real-time turbidity meter LA200, and the turbidity reached the threshold value of 0.1 within 30 minutes. The test result was considered positive. The test was conducted 20 times.
＜６＞カワヒバリガイ環境ＤＮＡのＰＣＲ検出およびＬＡＭＰ検出の比較（野外調査）
農業用水（枝下用水、愛知県豊田市）でそれぞれ約１ｋｍ離れた１０カ所の地点から１Ｌ採水し、簡易ろ過法（ガラス繊維ろ紙ＧＡ－５５、添加量１０ｍｇ、１５秒破砕、０．１８ｍｍメッシュシート）と従来法（環境ＤＮＡ調査・実験マニュアル）でろ過した。前述と同様の試薬を用いてＤＮＡを抽出し、リアルタイムＰＣＲ法およびＬＡＭＰ法で分析した。
<6> Comparison of PCR detection and LAMP detection of Kawahibari mussel environmental DNA (field survey)
1L of agricultural water (Edashita Water, Toyota City, Aichi Prefecture) was sampled from 10 locations approximately 1km apart from each other, and filtered using a simple filtration method (glass fiber filter paper GA-55, added amount 10mg, crushed for 15 seconds, 0.18mm). mesh sheet) and the conventional method (Environmental DNA Investigation/Experiment Manual). DNA was extracted using the same reagents as described above and analyzed by real-time PCR and LAMP.
＜７＞土壌懸濁水のDNA分析の比較
あらかじめＤＮＡ量が明らかなＬａｍｂｄａ ＤＮＡ（ＴＡＫＡＲＡ）を用いて、１０６ｃｏｐｉｅｓ／ｓｏｉｌに濃度調製して、黄色土および黒ぼく土０．５ｇに加えて混合した。これを１００ｍＬの滅菌水に加え、さらに懸濁させたガラス繊維５ｍｇ（１５秒破砕）を加えて良く撹拌し、図４に示すろ過分離を行う器具を用いてろ過し、回収したガラス繊維からＭｉｇｈｔｙＰｒｅｐ Ｒｅａｇｅｎｔ ｆｏｒ ＤＮＡ でＤＮＡを抽出した。同時に、市販の土壌ＤＮＡ抽出キットであるＮｕｃｌｅｏＳｐｉｎ Ｓｏｉｌ（ＴＡＫＡＲＡ）で添付のプロトコルに従ってＤＮＡを抽出した。それぞれの抽出ＤＮＡについて、リアルタイムＰＣＲを用いてＬａｍｂｄａ ＤＮＡ検出用のＰＣＲプライマー（Boulter et al,2016）によりｑＰＣＲ反応を行い、ＤＮＡ回収率を求めた。
Boulter N, Suarez FG, Schibeci S, Sunderland T, Tolhurst O, Hunter T, Hodge G, Handelsman D, Simanainen U, Hendriks E, Duggan K (2016) A simple, accurate and universal method for quantification of PCR. BMC Biotechnology: 16:27. doi:10.1186/s12896-016-0256-y
<7> Comparison of DNA analysis of soil suspension water Using Lambda DNA (TAKARA) whose DNA amount is known in advance, the concentration was adjusted to 10 6 copies/soil and mixed in addition to 0.5 g of yellow soil and black soil. did. Add this to 100 mL of sterilized water, add 5 mg of suspended glass fiber (crushed for 15 seconds), stir well, filter using the filtration separation device shown in Figure 4, and use MightyPrep from the recovered glass fiber. DNA was extracted using Reagent for DNA. At the same time, DNA was extracted using NucleoSpin Soil (TAKARA), a commercially available soil DNA extraction kit, according to the attached protocol. For each extracted DNA, a qPCR reaction was performed using real-time PCR using PCR primers for Lambda DNA detection (Boulter et al, 2016), and the DNA recovery rate was determined.
Boulter N, Suarez FG, Schibeci S, Sunderland T, Tolhurst O, Hunter T, Hodge G, Handelsman D, Simanainen U, Hendriks E, Duggan K (2016) A simple, accurate and universal method for quantification of PCR. BMC Biotechnology: 16:27. doi:10.1186/s12896-016-0256-y
＜８＞結果および考察
図５にガラス繊維（ＧＡ－５５）の破砕処理別の繊維長を示した。破砕時間が長いほど繊維が短くなること、および手もみが最も繊維が短いことが明らかとなった。なお、データは省略するが、ＧＡ－５５の他に、ＧＢ－１４０（Ａｄｖａｎｔｅｃ）、ＧＦ／Ａ（Ｗｈａｔｍａｎ）、ＧＦ／Ｄ（同）、ＧＦ／Ｆ（同）について、１５秒破砕後の繊維長を調査した結果、ＧＦ／Ｆの繊維が比較的長い傾向があったが、他には大きな差は見られなかった。
<8> Results and Discussion Figure 5 shows the fiber length of glass fiber (GA-55) according to the crushing treatment. It became clear that the longer the crushing time, the shorter the fibers, and that hand kneading produced the shortest fibers. Although the data is omitted, in addition to GA-55, GB-140 (Advantec), GF/A (Whatman), GF/D (same), and GF/F (same) have fibers after crushing for 15 seconds. As a result of examining the length, it was found that the fibers of GF/F tended to be relatively long, but no other major differences were observed.
図６にメッシュシート別のガラス繊維の捕捉率（％）の平均値を示した。１．０ｍｍのシートではガラス繊維がほぼ通過してしまい、捕捉率が低かった。またシートの目開きが小さいほど、多くのガラス繊維を捕捉できることが明らかとなった。しかし、手もみのガラス繊維は繊維が短すぎて、最小の０．０７ｍｍのシートでさえ流失する割合が高いことがわかった。 FIG. 6 shows the average value of the glass fiber capture rate (%) for each mesh sheet. With a 1.0 mm sheet, most of the glass fibers passed through, resulting in a low capture rate. It was also revealed that the smaller the sheet opening, the more glass fibers could be captured. However, it was found that the fibers of hand-kneaded glass fibers were too short, and even the smallest sheets of 0.07 mm were washed away at a high rate.
図７に池水１Ｌのろ過に要する時間（秒）の平均値を示した。シートの目開きが小さいほど、ろ過時間が長く、０．０７ｍｍのシートでは５秒破砕のガラス繊維以外は目詰まりして計測できなかった。図５および図６に示した結果から、ガラス繊維は手もみは適さず、５秒から６０秒破砕すると良いこと、およびシートは０．２８ｍｍから０．１８ｍｍが適していることがわかった。 FIG. 7 shows the average value of the time (seconds) required to filter 1 L of pond water. The smaller the opening of the sheet, the longer the filtration time, and in the case of a 0.07 mm sheet, all fibers other than glass fibers crushed for 5 seconds were clogged and could not be measured. From the results shown in FIGS. 5 and 6, it was found that hand kneading is not suitable for glass fibers, but it is better to crush them for 5 to 60 seconds, and that the sheet size is suitable for 0.28 mm to 0.18 mm.
図８にカワヒバリガイ環境ＤＮＡの定量結果（平均値）を示した。６０秒破砕したガラス繊維および０．０７ｍｍシートの組み合わせが最もＤＮＡ量が多く、また破砕時間が長い（繊維が短い）ほどＤＮＡ量が多い傾向がみられた。しかし、これまでの実験から０．０７ｍｍのシートでは目詰まりする可能性が高いこと、および０．２８ｍｍまたは０．１８ｍｍのシートでは６０秒破砕のガラス繊維の優位性が確認できないことから、ガラス繊維は５秒または１５秒破砕すると良いこと、およびシートは０．２８ｍｍから０．１８ｍｍが適していることがわかった。 Figure 8 shows the quantitative results (average value) of the environmental DNA of Kawahibari mussel. The combination of glass fiber and 0.07 mm sheet crushed for 60 seconds had the highest amount of DNA, and there was a tendency that the longer the crushing time (shorter fiber), the higher the amount of DNA. However, from previous experiments, it has been found that there is a high possibility of clogging with 0.07 mm sheets, and that the superiority of glass fibers crushed for 60 seconds cannot be confirmed with 0.28 mm or 0.18 mm sheets. It was found that crushing for 5 seconds or 15 seconds is good, and that the sheet size is suitable from 0.28 mm to 0.18 mm.
図９にガラス繊維の添加量ごとのＤＮＡ吸着効果の実験結果を示した。横軸はガラス繊維の添加量を、縦軸は吸着率の平均値を表している。添加量５０ｍｇの場合は平均で８３．７％のＤＮＡがガラス繊維に吸着されたことが明らかとなった。しかし、反復間のばらつきが大きく、差が認められたのは２０ｍｇと５０ｍｇの吸着率だけであり、その他では有意差はなかった。添加量を５０ｍｇとした場合は、その後に行うＭｉｇｈｔｙＰｒｅｐ Ｒｅａｇｅｎｔ ｆｏｒ ＤＮＡによるＤＮＡ抽出の工程において、試薬による反応が不十分となることが多かった。従って、本発明において添加するガラス繊維の量は、ＤＮＡ吸着率および抽出工程におけるハンドリングの観点から１０ｍｇ～２０ｍｇが適量であると考えられた。 FIG. 9 shows the experimental results of the DNA adsorption effect for each amount of glass fiber added. The horizontal axis represents the amount of glass fiber added, and the vertical axis represents the average adsorption rate. It was revealed that when the amount added was 50 mg, 83.7% of the DNA was adsorbed on the glass fiber on average. However, the variation between repetitions was large, and differences were observed only in the adsorption rates of 20 mg and 50 mg, and there were no significant differences in other cases. When the amount added was 50 mg, the reaction with the reagent was often insufficient in the subsequent DNA extraction step using MightyPrep Reagent for DNA. Therefore, it was considered that the appropriate amount of glass fiber to be added in the present invention is 10 mg to 20 mg from the viewpoint of DNA adsorption rate and handling in the extraction process.
表２に実験で使用したガラス繊維の種類、価格およびカワヒバリガイ飼育水中の環境ＤＮＡをリアルタイムＰＣＲおよびＬＡＭＰ法で分析した結果を示した。リアルタイムＰＣＲの値はＣｔ値を、ＬＡＭＰ法の値はＬＡＭＰ反応により生じた濁度が閾値０．１に達した時間（分）を表している。この結果、リアルタイムＰＣＲ法もＬＡＭＰ法も供試した１０種類のガラス繊維の全てＤＮＡが検出され、Ｃｔ値または反応時間（分）に大きな差はなかった。このことから、添加するガラス繊維のメーカーや規格を問わず、ＤＮＡ吸着に使用できると考えられた。 Table 2 shows the type and price of the glass fibers used in the experiment, and the results of analyzing the environmental DNA in the water for breeding snails using real-time PCR and LAMP methods. The value of real-time PCR represents the Ct value, and the value of the LAMP method represents the time (minutes) at which the turbidity generated by the LAMP reaction reached the threshold value of 0.1. As a result, DNA was detected in all 10 types of glass fibers tested using both the real-time PCR method and the LAMP method, and there was no significant difference in Ct value or reaction time (minutes). From this, it was thought that it could be used for DNA adsorption regardless of the manufacturer or standard of the added glass fiber.
図１０に簡易ろ過法と従来法でろ過抽出したカワヒバリガイ飼育水中の環境ＤＮＡについて、リアルタイムＰＣＲ法およびＬＡＭＰ法で分析した結果を示した。簡易ろ過法で調製したＤＮＡをＬＡＭＰ分析した結果、２０検体中１９検体が陽性となり、陽性率が最も高かった。このことから簡易ろ過法でろ過抽出したＤＮＡはＬＡＭＰ分析に適していると考えられた。 Figure 10 shows the results of analysis using the real-time PCR method and the LAMP method for environmental DNA in the water for cultivating snails that was filtered and extracted using the simple filtration method and the conventional method. As a result of LAMP analysis of DNA prepared by a simple filtration method, 19 out of 20 samples were positive, which was the highest positive rate. From this, it was considered that DNA filtered and extracted by the simple filtration method is suitable for LAMP analysis.
表３は、実施例における農業用水中のカワヒバリガイの環境ＤＮＡをリアルタイムＰＣＲ法およびＬＡＭＰ法で分析し、陽性率を比較した表である。簡易ろ過法で調製したＤＮＡはＬＡＭＰ分析では全て陽性となり、従来法で抽出したＤＮＡをＰＣＲ分析した場合と結果が完全に一致した。このことから前述の室内実験と同様に野外調査でも、簡易ろ過法でろ過抽出したＤＮＡはＬＡＭＰ分析に適していることが実証された。 Table 3 is a table comparing the positive rates of the environmental DNA of the snails in agricultural water in Examples, analyzed by real-time PCR method and LAMP method. The DNA prepared by the simple filtration method was all positive in the LAMP analysis, and the results were completely consistent with the PCR analysis of DNA extracted by the conventional method. From this, the field investigation as well as the above-mentioned indoor experiment demonstrated that DNA filtered and extracted using the simple filtration method is suitable for LAMP analysis.
表４は、実施例における土壌に添加したＬａｍｂｄａ ＤＮＡを市販のキットとガラス繊維を用いた簡易ろ過法で抽出して、ＰＣＲ法で分析した結果を示した表である。簡易ろ過法で調製した場合のＤＮＡ回収率は、黄色土では３．８～４．２％、黒ぼく土では０．７～１．５％だったのに対し、ＮｕｃｌｅＳｐｉｎ ｓｏｉｌ抽出のＤＮＡ回収率は、それぞれ約１２．３％および１１％だった。簡易ろ過法のＤＮＡ回収率は市販のＤＮＡ抽出キットの約１／３～１／１６と低く、Ｃｔ値が３～４サイクル遅くなるものの、ＤＮＡ増幅産物のＴｍ値は市販抽出キットとほぼ一致しており、簡易ろ過法で土壌中のＤＮＡを抽出することは可能と考えられた。 Table 4 is a table showing the results of extracting Lambda DNA added to the soil in Examples using a simple filtration method using a commercially available kit and glass fiber, and analyzing it using a PCR method. The DNA recovery rate when prepared using a simple filtration method was 3.8-4.2% for yellow soil and 0.7-1.5% for black soil, whereas the DNA recovery rate for NucleSpin soil extraction was were approximately 12.3% and 11%, respectively. Although the DNA recovery rate of the simple filtration method is about 1/3 to 1/16 of that of commercially available DNA extraction kits, and the Ct value is 3 to 4 cycles slower, the Tm value of the DNA amplification product is almost the same as that of commercially available extraction kits. Therefore, it was considered possible to extract DNA from soil using a simple filtration method.
以上の実験結果から、ガラス繊維に水中のＤＮＡを吸着させてメッシュシート等で回収するという本発明の方法は、工程が極めて簡単であるだけでなく、その後のＤＮＡ抽出にＭｉｇｈｔｙＰｒｅｐ Ｒｅａｇｅｎｔ ｆｏｒ ＤＮＡに代表される簡易なＤＮＡ抽出試薬を利用でき、さらにこうして抽出したＤＮＡは遺伝子診断法として最も一般的なＰＣＲ法だけでなく、むしろＬＡＭＰ法での検出に適していることが明らかとなった。実施例では、環境水および土壌水を対象としたが、対象はこれらだけにとどまるものではない。例えば、空中に浮遊、飛散しているウイルス、花粉、胞子、微小昆虫、など、水に溶解されれば、その後の工程は環境水と同様に扱うことができると考えられる。従って、本発明は環境中の様々な生物相の分析に活用が期待できる。 From the above experimental results, we found that the method of the present invention, in which DNA in water is adsorbed onto glass fibers and recovered using a mesh sheet, etc., not only has an extremely simple process, but also uses MightyPrep Reagent for DNA for subsequent DNA extraction. It has become clear that a simple DNA extraction reagent can be used, and that the DNA thus extracted is suitable for detection not only by the PCR method, which is the most common genetic diagnostic method, but also by the LAMP method. In the examples, environmental water and soil water were targeted, but the targets are not limited to these. For example, if airborne viruses, pollen, spores, microscopic insects, etc. are dissolved in water, they can be treated in the same way as environmental water in subsequent processes. Therefore, the present invention can be expected to be utilized for analysis of various biota in the environment.
１ キャップ付きサンプリングバッグ
２ ホース
３ シリコン栓
４ 塩化ビニル製のアダプタ
５ メッシュシート
６ 塩化ビニル製のアダプタ
１１ 吸引カップ
１２ メッシュシート
１３ 吸引瓶
1 Sampling bag with cap 2 Hose 3 Silicone plug 4 PVC adapter 5 Mesh sheet 6 PVC adapter 11 Suction cup 12 Mesh sheet 13 Suction bottle
Claims (9)
微細な編み目構造を有するシートの目開きが、０．１８ｍｍ～０．２８ｍｍであり、
核酸を吸着させるためのガラス繊維の繊維長の分布が、繊維長５０μｍ以下が１０～６５％、繊維長５１μｍ以上３００μｍ以下が３０～６０％、繊維長３０１μｍ以上が５～４０％であり、
検体が環境水である、
方法。 Nucleic acids in the specimen are collected by adsorbing the nucleic acids in the specimen onto glass fibers suspended in water, and collecting the glass fibers to which the nucleic acids have been adsorbed by filtering and separating them through a sheet with a fine mesh structure. A method of collecting
The opening of the sheet having a fine mesh structure is 0.18 mm to 0.28 mm,
The fiber length distribution of the glass fiber for adsorbing nucleic acids is such that 10 to 65% have a fiber length of 50 μm or less, 30 to 60% have a fiber length of 51 μm or more and 300 μm or less, and 5 to 40% have a fiber length of 301 μm or more.
The sample is environmental water,
Method.
（ａ）液体を流入するための開口部および液体を排出するための開口部を有する袋、及び
（ｂ）前記の液体を排出するための開口部に設置された、２つの円筒管に挟まれた微細な編み目構造を有するシート、
を有するサンプリングバッグを使用して実施する、請求項１に記載の方法。 The process of recovering glass fibers to which nucleic acids have been adsorbed is carried out by filtering and separating them through a sheet with a fine mesh structure.
(a) a bag having an opening for the inflow of liquid and an opening for the evacuation of the liquid; and (b) sandwiched between two cylindrical tubes placed in the opening for the evacuation of said liquid. A sheet with a fine mesh structure,
2. The method of claim 1, wherein the method is carried out using a sampling bag having:
液体を流入するための開口部および液体を排出するための開口部を有する吸引カップと、前記の液体を排出するための開口部から排出される液体を受けるための容器とを含み、前記吸引カップと前記容器との間に、微細な編み目構造を有するシートが設置されているＤＮＡろ過器具を使用して実施する、請求項１に記載の方法。 The process of recovering glass fibers to which nucleic acids have been adsorbed is carried out by filtering and separating them through a sheet with a fine mesh structure.
a suction cup having an opening for entering liquid and an opening for discharging liquid; and a container for receiving liquid discharged from the opening for discharging said liquid; 2. The method according to claim 1, wherein the method is carried out using a DNA filtration device in which a sheet having a fine mesh structure is installed between the DNA filtration device and the container.
回収した核酸を用いて、ＬＡＭＰ法によりＤＮＡの増幅を行う工程、
を含む、環境ＤＮＡの検出方法。 A step of recovering nucleic acid in a specimen by the method according to any one of claims 1 to 8 , and a step of amplifying DNA by the LAMP method using the recovered nucleic acid,
A method for detecting environmental DNA.
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| JP2005218321A (en) | 2004-02-03 | 2005-08-18 | Gifu Prefecture | Nucleic acid isolation method and nucleic acid binding carrier |
| JP2005304504A (en) | 1993-08-30 | 2005-11-04 | Promega Corp | Nucleic acid purification composition and method |
| JP2011502536A (en) | 2007-11-16 | 2011-01-27 | イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー | Method for detecting and / or analyzing yeast and mold in a filterable liquid |
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| JP2005304504A (en) | 1993-08-30 | 2005-11-04 | Promega Corp | Nucleic acid purification composition and method |
| JP2005218321A (en) | 2004-02-03 | 2005-08-18 | Gifu Prefecture | Nucleic acid isolation method and nucleic acid binding carrier |
| JP2011502536A (en) | 2007-11-16 | 2011-01-27 | イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー | Method for detecting and / or analyzing yeast and mold in a filterable liquid |
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