JP6057175B2 - A method for decomposing and removing DNA at high speed without leaking contamination using a non-degassing high-temperature high-pressure steam treatment apparatus - Google Patents
A method for decomposing and removing DNA at high speed without leaking contamination using a non-degassing high-temperature high-pressure steam treatment apparatus Download PDFInfo
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Description
本発明は、周辺環境に汚染を拡散することなく、より高速に鋳型ＤＮＡを分解除去する方法に関する。 The present invention relates to a method for decomposing and removing template DNA at a higher speed without diffusing contamination into the surrounding environment.
ＤＮＡの塩基配列を解読したり、特定の配列を有するＤＮＡのコピー数を定量する方法としては、特定の塩基配列を有するＤＮＡの一部の領域をＰＣＲ法等で数千億のコピーであるＤＮＡの断片に増幅し、その増幅過程または増幅産物を解析するという方法が一般的である。しかし、ＰＣＲで得られたＤＮＡの断片はそれ自体が鋳型としての活性を有しているため、これらがサンプルや試薬等に混入すると再びＰＣＲの鋳型となって増幅される可能性がある。近年ＰＣＲ法の普及に伴い、ＰＣＲ鋳型活性を有するＤＮＡ断片を多量に含む医療・実験廃棄物が発生し、不適切な処置によりフォルス・ポジティブを発生させることが深刻な問題になっている。一般的にＰＣＲの増幅産物を裸で扱う実験室等はそのＤＮＡの断片で汚染されており、正確な定量や診断が出来なくなるという問題を抱えている。 As a method of deciphering the base sequence of DNA or quantifying the number of copies of DNA having a specific sequence, a partial region of DNA having a specific base sequence can be obtained by using a PCR method, etc. In general, a method of amplifying the fragment and analyzing the amplification process or amplification product is generally used. However, since DNA fragments obtained by PCR themselves have activity as a template, they may be amplified again as a PCR template if they are mixed into a sample or reagent. In recent years, with the spread of the PCR method, medical / experimental waste containing a large amount of DNA fragments having PCR template activity is generated, and it has become a serious problem to generate false positives by inappropriate treatment. In general, laboratories that handle PCR amplification products naked are contaminated with DNA fragments and have a problem that accurate quantification and diagnosis cannot be performed.
ＤＮＡの鋳型活性の不活化に関しては、従来の高温高圧水蒸気処理装置（オートクレーブ）による１２１℃２０分間の処理では十分な不活化が出来ないということが経験的に明らかであり、多くの報告もなされている。オートクレーブに関しては、本来細菌や細菌の芽胞を死滅させるために用いられてきた装置であり、歴史的経験則から飽和の水蒸気（即ち空気を含まない水蒸気）により１２１℃で１５−２０分間処理するという条件が採用されている。飽和水蒸気を用いる理由としては細菌や芽胞に対する殺傷性が高いという理由のほかに、圧力容器内から空気を排除することにより偏りなく１２１℃の雰囲気を醸成するという目的がある。そのため加熱から殺菌操作の過程で断続的に空気が混じった水蒸気を弁を通じて装置外に放出するという行為が繰り返されることになるが、この空気と水蒸気の混合物には微量ながらＤＮＡの断片が混入する。このため、ＰＣＲ産物等の大量の鋳型ＤＮＡを含む廃棄物をオートクレーブで処理すること自体が実験室環境の汚染やフォルス・ポジティブの原因になっていた。
また、ＤＮＡの鋳型活性を不活化するためには化学処理、紫外線による処理、γ線による処理などが試みられてきたが、いずれも完全に鋳型活性を除くことは難しいとされてきた（特許文献１，非特許文献１〜５等参照）。一方でＤＮase等の酵素を利用することがＤＮＡの分解除去には最も有効であるが、酵素そのものが高価であること、全ての実験器具や試薬類、廃棄物類を処理できるフォーマットの技術ではないことから、特定の場合を除き、あまり現実的なＤＮＡの分解除去方法とは言えなかった。
Regarding the inactivation of the template activity of DNA, it is empirically clear that the inactivation at 121 ° C. for 20 minutes by a conventional high-temperature and high-pressure steam treatment apparatus (autoclave) cannot be performed sufficiently, and many reports have been made. ing. The autoclave is an apparatus that has been originally used to kill bacteria and bacterial spores. According to a historical rule of thumb, it is treated with saturated water vapor (ie, water-free water vapor) at 121 ° C. for 15-20 minutes. Conditions are adopted. The reason why saturated water vapor is used is that, in addition to the high killing ability against bacteria and spores, there is a purpose of fostering an atmosphere of 121 ° C. without bias by eliminating air from the inside of the pressure vessel. For this reason, the action of intermittently mixing water mixed with air during the process of heating and sterilization is repeatedly released to the outside of the apparatus through the valve. However, a small amount of DNA fragments are mixed in this mixture of air and water vapor. . For this reason, treating waste containing a large amount of template DNA such as PCR products with an autoclave itself caused contamination of the laboratory environment and false positives.
In addition, in order to inactivate the template activity of DNA, chemical treatment, treatment with ultraviolet rays, treatment with γ-rays, etc. have been tried, but it has been difficult to completely remove the template activity (patent document). 1, Non-Patent
既存のオートクレーブを用いてＰＣＲ産物等のＤＮＡ断片を高温高圧水蒸気処理する場合には次の１）〜３）の問題点が存在する。
１）オートクレーブはそもそもがＤＮＡを分解除去するための技術では無いので、一般的に用いられる条件では十分なＤＮＡの分解処理ができない。
２）オートクレーブは加熱・高温高圧処理の過程で圧力容器内を飽和の水蒸気で置換するため大量の空気と水蒸気を排出するシステムを採用している。このため排気を通じて処理装置内から未処理のＤＮＡ断片が漏出し、周囲を汚染する。
３）上記の排気という操作を行わないと、処理装置内部に高温の水蒸気の層と低温の空気の層が生じてしまい、局所的に十分な処理条件が担保できなくなる。
本発明が解決しようとする課題は、上記１）〜３）に列挙した、従来のオートクレーブの原理・構造・使用方法に起因して生ずる問題点を解消する点にあり、鋳型活性を有するＤＮＡ断片を含む廃棄物を、周辺環境を汚染することなく不活性化することにある。
When DNA fragments such as PCR products are subjected to high-temperature and high-pressure steam treatment using an existing autoclave, the following problems 1) to 3) exist.
1) Since an autoclave is not a technique for decomposing and removing DNA in the first place, a sufficient DNA decomposing process cannot be performed under generally used conditions.
2) The autoclave employs a system that discharges a large amount of air and water vapor in order to replace the inside of the pressure vessel with saturated water vapor in the process of heating and high temperature and pressure. For this reason, unprocessed DNA fragments leak from the processing apparatus through the exhaust, and the surroundings are contaminated.
3) If the above-described operation of exhaust is not performed, a high-temperature water vapor layer and a low-temperature air layer are generated inside the processing apparatus, and sufficient processing conditions cannot be secured locally.
The problem to be solved by the present invention is to eliminate the problems caused by the principle, structure and method of use of conventional autoclaves listed in 1) to 3) above, and a DNA fragment having template activity. It is intended to inactivate waste containing no contamination of the surrounding environment.
これらを解決するために、鋳型ＤＮＡを外部に漏らさずに処理する方法を鋭意研究した結果、ＤＮＡを含む水溶液を空気（または酸素を含む気体）と共に容器に密閉した状態で高温高圧水蒸気処理することにより、鋳型ＤＮＡを外部に漏らさず、かつ、空気または酸素が共存することによってより高速に鋳型ＤＮＡを分解除去できるという事実を見出し、排気中に未処理のＤＮＡ断片を放出させずに高温高圧水蒸気処理を行うことに関してその有効性を確認し、また、空気（または酸素を含む気体）と密閉した容器中で高温高圧水蒸気処理を行うことがよりＤＮＡ断片の分解処理には有効であるという新たな知見を得た。
具体的には、以下の１〜３の手順に従う。
（１）ＰＣＲ増幅産物等のＤＮＡ断片を含む水溶液を空気（または酸素を含む気体）と共に容器に密閉する。
（２）通常のオートクレーブ内に上記密閉容器を配置し、１２１℃で８０分程度保持し、密閉容器内で高温高圧水蒸気処理を行う。
（３）室温まで冷却後に密閉容器を開け、処理後の残存物を取り出す。
上記の方法で処理するＤＮＡを含む水溶液は１０マイクロリットル程度、上記の操作に用いる密閉容器は２ミリリットル程度のものが望ましい。
In order to solve these problems, as a result of earnest research on a method for treating template DNA without leaking to the outside, high-temperature and high-pressure steam treatment is performed in a state where an aqueous solution containing DNA is sealed in a container together with air (or a gas containing oxygen). Thus, the fact that the template DNA can be decomposed and removed at higher speed without leaking the template DNA to the outside and coexisting with air or oxygen is found, and high temperature and high pressure water vapor is released without releasing untreated DNA fragments into the exhaust gas. The effectiveness of the treatment is confirmed, and a new high-temperature high-pressure steam treatment in a sealed container with air (or a gas containing oxygen) is more effective for decomposing DNA fragments. Obtained knowledge.
Specifically, the following
(1) An aqueous solution containing a DNA fragment such as a PCR amplification product is sealed in a container together with air (or a gas containing oxygen).
(2) The above sealed container is placed in a normal autoclave, kept at 121 ° C. for about 80 minutes, and subjected to high-temperature and high-pressure steam treatment in the sealed container.
(3) After cooling to room temperature, the sealed container is opened and the treated residue is taken out.
The aqueous solution containing DNA to be treated by the above method is preferably about 10 microliters, and the sealed container used for the above operation is preferably about 2 milliliters.
容量が大きな密閉容器内では内部の温度の不均衡や水蒸気と空気の偏在が生じ、十分な処理効果が得られない可能性がある。より理想的には、発明を実施するための形態として後記するように、高温高圧で水蒸気処理する装置そのものを密閉型にし、ＤＮＡ断片を含む水蒸気を外部に漏らさない一方で大容量の空気と水蒸気の混合気体によって高温高圧処理をすることが簡便で確実に鋳型ＤＮＡを分解除去するために有用である。 In a closed container with a large capacity, internal temperature imbalance and water vapor and air may be unevenly distributed, and a sufficient treatment effect may not be obtained. More ideally, as will be described later as a mode for carrying out the invention, the apparatus for performing steam treatment at high temperature and high pressure is hermetically sealed so that water containing DNA fragments is not leaked to the outside, while a large volume of air and steam. It is useful to perform the high-temperature and high-pressure treatment with the mixed gas in order to easily and reliably decompose and remove the template DNA.
本発明によれば、鋳型活性を有するＤＮＡ断片を周辺環境に漏らさず、確実に分解除去することが出来る。従来に比べて実験室等の環境をよりクリーンに保つことにより、フォルス・ポジティブの発生を回避しつつより精密なＤＮＡ断片の定量や塩基配列の解析を可能にする。
また、従来は、１０の１１乗コピー程度（通常のＰＣＲで生じる増幅産物量）のＰＣＲ鋳型活性を完全に除去するためには１２１℃で１００分間以上の処理時間が必要であり、ＰＣＲの廃棄物等を処理する技術としては改善が必要であったが、本発明によれば、ＤＮＡを含む水溶液を完全に密閉して空気が外部の水蒸気に置換されないような条件で１２１℃の高温高圧水蒸気処理を行うことにより、水溶液中のＤＮＡは分解除去することが出来、さらに所要時間も８０分程度に短縮される。
また、従来のオートクレーブでは高圧容器内を１２１℃の水蒸気で均一に満たすために断続的に排気を行って空気を除去していたが、発明を実施するための形態として後記する高温高圧水蒸気処理装置を用いて１２１℃で８０分程度の処理を行えば、小さな容器に封入できるＤＮＡ溶液だけではなく、ＤＮＡが付着した器具やグローブ、紙タオル等の廃棄物、大量の廃液なども周囲に汚染を発生することなく処理することが可能になる。
According to the present invention, a DNA fragment having template activity can be reliably decomposed and removed without leaking to the surrounding environment. By keeping the laboratory environment cleaner than before, it is possible to more accurately quantify DNA fragments and analyze base sequences while avoiding false positives.
Conventionally, in order to completely remove the PCR template activity of about 10 11 copies (the amount of amplification product generated by normal PCR), a treatment time of at least 100 minutes at 121 ° C. is required. However, according to the present invention, the high-temperature and high-pressure steam at 121 ° C. is used under such a condition that the aqueous solution containing DNA is completely sealed and the air is not replaced by external steam. By performing the treatment, the DNA in the aqueous solution can be decomposed and removed, and the required time is further shortened to about 80 minutes.
Further, in the conventional autoclave, the air was removed by intermittently exhausting in order to uniformly fill the inside of the high-pressure vessel with water vapor at 121 ° C., but the high-temperature high-pressure steam treatment apparatus described later as a form for carrying out the invention If the treatment is performed at 121 ° C. for about 80 minutes using the, the not only DNA solution that can be sealed in a small container, but also wastes such as DNA attached instruments, gloves, paper towels, etc. It becomes possible to process without generating.
実際の鋳型活性を有するＤＮＡ断片を含む廃棄物は、小容量の容器に密閉できる形状のものには限定されない。したがって、より大きな容量で非脱気の高温高圧水蒸気処理を行うことが望ましい。具体的には、既存のオートクレーブ等の脱気式高温高圧水蒸気処理装置では無く、内部からの蒸気を外部に排出させず、かつ、一様に空気（または酸素）を含む水蒸気で高温高圧に処理する装置を利用し、以下の１〜５の手順に従って処理することにより、様々なフォーマットの廃棄物中に含まれる鋳型ＤＮＡを高効率かつ簡便・確実に分解することが可能になる。
（１）高温高圧水蒸気処理装置内に剥き出しの廃棄物や非密閉の容器に入れた廃液類を入れる。必要に応じてステンレス製のバスケットやバケツ、オートクレーブ用のプラスチック・バッグなどを利用してまとめて処理ができる。
（２）上述の鋳型ＤＮＡを含む廃棄物等と水と空気を処理室内に密閉し、排気を行わずにボイラーの加熱を行う。
（３）このとき処理室内では高温の水蒸気の層と低温の空気の層が生じることがないようにサーキュレーターまたは水蒸気と空気の対流を制御する内部の構造等によって温度と空気・水蒸気の組成の均一化をはかる。
（４）上記のようにして発生させた１２１℃の空気と水蒸気の混合気体中で廃棄物や廃液類を８０分程度処理する。
（５）室温まで冷却後に処理装置を開け、処理後の廃棄物や廃液類を取り出す。
The waste containing the DNA fragment having actual template activity is not limited to a shape that can be sealed in a small-capacity container. Therefore, it is desirable to perform non-degassing high-temperature high-pressure steam treatment with a larger capacity. Specifically, it is not a degassing high-temperature and high-pressure steam treatment device such as an existing autoclave, and steam from the inside is not discharged to the outside, and it is uniformly treated at high temperature and pressure with steam containing air (or oxygen). By using the apparatus to perform the process according to the following
(1) Place bare waste or waste liquid in a non-sealed container in a high-temperature, high-pressure steam treatment apparatus. If necessary, it can be processed collectively using stainless steel baskets and buckets, plastic bags for autoclaves, etc.
(2) The waste containing the above template DNA, water and air are sealed in the treatment chamber, and the boiler is heated without exhausting.
(3) At this time, the temperature and the composition of air and water vapor are made uniform by a circulator or an internal structure that controls the convection of water vapor and air so that a high temperature water vapor layer and a low temperature air layer are not generated in the processing chamber. Measure.
(4) Waste and waste liquids are treated for about 80 minutes in a mixed gas of 121 ° C. air and water vapor generated as described above.
(5) After cooling to room temperature, the processing apparatus is opened, and the processed waste and waste liquid are taken out.
以下本発明の実施例を示すが、本発明はこれら実施例により限定されるものではない。
［実施例１］
（１）ｐＵＣ１９プラスミドから「５’−ＴＧＧＣＧＴＡＡＴＣＡＴＧＧＴＣＡＴＡＧＣＴＧ」と「５’−ＴＧＣＡＴＧＣＣＴＧＣＡＧＧＴＣＧＡＣＴＣ」の配列を有する一対のプライマーを用いて２,６８２塩基対のＤＮＡ断片をＰＣＲ増幅した。８.７１×１０10コピー／μＬの濃度で当該ＤＮＡ鋳型を含む水溶液を得、この水溶液をモデルＰＣＲ廃棄物とした。
（２）モデルＰＣＲ廃棄物の水溶液を１０μＬずつ、ポリプロピレン製のマイクロチューブ（アシストチューブ型番７２．６９４．１００Ｓ）４本に分注し、エチレン−プロピレンゴムのＯ−リングを介して専用のネジ口式の蓋で密栓した。また、分注前後のチューブの重量を０.０１μｇの桁で測定し、正確な溶液の分注量を確認した。
（３）密閉状態のままオートクレーブ内に設置し、２０分、４０分、または１２０分間１２１℃で処理した。
（４）３５℃まで冷却後にチューブを取り出し、それぞれ重量を測定して初期サンプル量の２倍容になるようにＴＥバッファーで希釈した。これを３μＬ電気泳動にかけた。
（５）残りのサンプルをさらに１０倍容（トータル２０倍容）に希釈して、そのうち１μＬ×２を鋳型活性の定量に用いた。
（６）鋳型活性の定量は定量ＰＣＲにて、８４塩基対の領域を増幅するプライマー（５’−ＣＴＧＴＣＧＴＧＣＣＡＧＣＴＧＣＡＴＴＡと５’−ＧＡＧＣＧＡＧＧＡＡＧＣＧＧＡＡＧＡＧ）およびその領域の増幅を検出するためのＴａｑＭａｎプローブ（５’ＦＡＭ−ＡＴＣＧＧＣＣＡＡＣＧＣＧＣＧＧＧ−３’ＴＡＭＲＡ）と、アプライドバイオシステムズ社の７９００ＨＴシーケンス・ディテクション・システムを使用して行った。
図１は定量ＰＣＲの増幅曲線で、Ｓ字の立ち上がりが生じるサイクルが左寄りで早いほどより多くの鋳型活性を検出したことになる。図中に示した通り、無処理のものに対して１２１℃で２０分、４０分と処理することにより鋳型活性が減少してゆくことがわかる。１２０分の処理の後には完全に鋳型活性が失われ、Ｓ字の増幅曲線は描かれなくなる。標準サンプルを用いて値付けした結果、１本のチューブに存在した鋳型の量はそれぞれ２０分処理後が５.７４×１０9コピー、４０分処理後が５.１８×１０2コピー、そして１２０分処理後がゼロコピーであった。以上の結果より、飽和水蒸気に曝されず、容器の中に密閉されたＤＮＡ溶液であっても、長時間処理することによりＰＣＲの鋳型活性を除去することができた。一方、同サンプルを電気泳動して臭化エチジウムで染色したものが図２である。第２レーンが無処理、第３レーンが２０分、第４レーンが４０分、第５レーンが１２０分処理後のサンプルであるが、ＤＮＡは１２１℃の処理で速やかに低分子化され、さらに臭化エチジウムで染色されない形状にまで変換されてゆくことが観察された。また、オートクレーブの底に残った水や排出された水蒸気から鋳型活性の増加は検出されなかった。
Examples of the present invention are shown below, but the present invention is not limited to these examples.
[Example 1]
(1) A DNA fragment of 2,682 base pairs was PCR amplified from the pUC19 plasmid using a pair of primers having the sequences of “5′-TGGGCGATATCATGGTCATAGCTG” and “5′-TGCATGCCTGCAGGGTCACTACT”. An aqueous solution containing the DNA template at a concentration of 8.71 × 10 10 copies / μL was obtained, and this aqueous solution was used as model PCR waste.
(2) Dispense 10 μL of model PCR waste solution into four polypropylene microtubes (assist tube model number 72.694.100S) and use dedicated screw caps via O-rings of ethylene-propylene rubber. Sealed with a formula lid. In addition, the weight of the tube before and after dispensing was measured with an order of 0.01 μg, and the exact amount of solution dispensed was confirmed.
(3) It set in the autoclave with airtight condition, and processed at 121 degreeC for 20 minutes, 40 minutes, or 120 minutes.
(4) After cooling to 35 ° C., the tubes were taken out, each weighed and diluted with TE buffer so as to be twice the initial sample volume. This was subjected to 3 μL electrophoresis.
(5) The remaining sample was further diluted to 10 volumes (20 volumes in total), and 1 μL × 2 was used for quantification of template activity.
(6) Template activity is determined by quantitative PCR using primers (5′-CGTGCTGGCCACGTGCATTA and 5′-GAGCGAGAGACGCGGAAGAG) for amplifying an 84 base pair region and a TaqMan probe (5′FAM−) for detecting the amplification of the region. ATCGGCCAACCGCGGGGG-3′TAMRA) and the 7900HT Sequence Detection System from Applied Biosystems.
FIG. 1 is an amplification curve of quantitative PCR, and the more the template activity is detected, the faster the cycle in which the S-shaped rise occurs to the left. As shown in the figure, it can be seen that the template activity decreases by treating the non-treated sample at 121 ° C. for 20 minutes and 40 minutes. After 120 minutes of treatment, template activity is completely lost and no S-shaped amplification curve is drawn. As a result of pricing using standard samples, the amount of template present in one tube was 5.74 × 10 9 copies after 20 minutes, 5.18 × 10 2 copies after 40 minutes, and 120, respectively. Zero copy after minute processing. From the above results, even if the DNA solution was not exposed to saturated water vapor and sealed in a container, the template activity of PCR could be removed by treating for a long time. On the other hand, FIG. 2 shows the sample electrophoresed and stained with ethidium bromide. The second lane was untreated, the third lane was 20 minutes, the fourth lane was 40 minutes, and the fifth lane was treated for 120 minutes, but the DNA was rapidly depolymerized by treatment at 121 ° C. It was observed that it was transformed into a shape that was not stained with ethidium bromide. Further, no increase in template activity was detected from the water remaining at the bottom of the autoclave or the discharged water vapor.
［比較例１］
（１）実施例１と同じモデルＰＣＲ廃棄物を使用した。
（２）モデルＰＣＲ廃棄物の水溶液を１０μＬずつ、ポリプロピレン製のマイクロチューブ（アシストチューブ型番７２．６９４．１００Ｓ）６本に分注し、エチレン−プロピレンゴムのＯ−リングを介して専用のネジ口式の蓋で密栓した。また、分注前後のチューブの重量を０.０１μｇの桁で測定し、正確な溶液の分注量を確認した。
（３）チューブ３本を密閉状態のまま、また、他のチューブ３本を蓋をせずに完全に気相が飽和の水蒸気と置換する状態でオートクレーブ内に設置し、４０分間１２１℃で処理した。
（４）３５℃まで冷却後にチューブを取り出し、それぞれ重量を測定して初期サンプル量の１００倍容になるようにＴＥバッファーで希釈した。そのうち１μＬ×２を鋳型活性の定量に用いた。
（５）鋳型活性の定量は実施例１と同様に行った。
図３はチューブ内に密閉して高温高圧の処理を行った結果の増幅曲線であるが、３本のチューブのうち２本では完全に鋳型活性が失われている。残りの１本では鋳型活性が残存しているが、チューブ１本当たり１.７４×１０4コピー程度の活性であることから、初期添加量に対して１０7分の１程度にまで鋳型活性が減少していることが分かる。一方、蓋をせずに飽和水蒸気で置換して処理した３本に関しては図４に示す通り全てのチューブで鋳型活性の残留が見られた。３本の平均値で５.９９×１０8コピー程度の活性が残ったことから、処理前の１０００分の１程度の活性が除去されずに残っている。なお、本検討を行うにあたり、実験室を汚染しないためにオートクレーブの排気は全て氷冷したプラスチック・バッグに回収して処分した。
[Comparative Example 1]
(1) The same model PCR waste as in Example 1 was used.
(2) Dispense 10 μL of model PCR waste solution into 6 polypropylene microtubes (assist tube model number 72.694.100S) and use dedicated screw caps through O-rings of ethylene-propylene rubber. Sealed with a formula lid. In addition, the weight of the tube before and after dispensing was measured with an order of 0.01 μg, and the exact amount of solution dispensed was confirmed.
(3) Place the 3 tubes in a sealed state and place the other 3 tubes in the autoclave in a state where the gas phase is completely replaced with saturated water vapor without closing the lid, and process at 121 ° C for 40 minutes. did.
(4) After cooling to 35 ° C., the tubes were taken out, each weighed and diluted with TE buffer so as to be 100 times the initial sample volume. Of these, 1 μL × 2 was used for quantification of template activity.
(5) The template activity was quantified in the same manner as in Example 1.
FIG. 3 is an amplification curve as a result of sealing in a tube and performing a high-temperature and high-pressure treatment, but the template activity is completely lost in two of the three tubes. Although template activity in the remaining one remaining, since it is the activity of one per 1.74 × 10 4 as copy, tube and mold activity to about one 10 7 minutes for the initial amount It turns out that it has decreased. On the other hand, as shown in FIG. 4, the remaining template activity was observed in all three tubes treated with saturated steam without using a lid. Since about 5.99 × 10 8 copies of activity remained with the average value of the three, about 1/1000 of the activity before the treatment remained without being removed. In this study, all the exhaust from the autoclave was collected in an ice-cooled plastic bag for disposal so as not to contaminate the laboratory.
［比較例２］
（１）実施例１と同じモデルＰＣＲ廃棄物を使用した。
（２）モデルＰＣＲ廃棄物の水溶液を１０μＬずつ、ポリプロピレン製のマイクロチューブ（アシストチューブ型番７２．６９４．１００Ｓ）６本に分注し、エチレン−プロピレンゴムのＯ−リングを介して専用のネジ口式の蓋で密栓した。また、分注前後のチューブの重量を０.０１μｇの桁で測定し、正確な溶液の分注量を確認した。
（３）チューブ３本の気相を純窒素で置換して密閉した。また他のチューブ３本の気相をエージレス（三菱ガス化学）を用いて酸素を除いた空気で置換して密閉した。オートクレーブ内に設置し、４０分間１２１℃で処理した。
（４）３５℃まで冷却後にチューブを取り出し、それぞれ重量を測定して初期サンプル量の１００倍容になるようにＴＥバッファーで希釈した。そのうち１μＬ×２を鋳型活性の定量に用いた。
（５）鋳型活性の定量は実施例１と同様に行った。
図５、図６はそれぞれ気相を純窒素、または、酸素を除いた空気と密閉して高温高圧処理を行った後のサンプルの増幅曲線であるが、いずれもしっかりとしたＳ字の曲線が観察され、比較例１の図３（酸素を含む空気と密閉した場合）と比較して、鋳型活性の除去が阻害されていることが分かる。以上の観察から、酸素が共存する状態での高温高圧水蒸気処理がＤＮＡの分解に有効であることが確認された。
[Comparative Example 2]
(1) The same model PCR waste as in Example 1 was used.
(2) Dispense 10 μL of model PCR waste solution into 6 polypropylene microtubes (assist tube model number 72.694.100S) and use dedicated screw caps through O-rings of ethylene-propylene rubber. Sealed with a formula lid. In addition, the weight of the tube before and after dispensing was measured with an order of 0.01 μg, and the exact amount of solution dispensed was confirmed.
(3) The gas phase of the three tubes was replaced with pure nitrogen and sealed. Further, the gas phase of the other three tubes was replaced with air from which oxygen was removed using AGELESS (Mitsubishi Gas Chemical) and sealed. Placed in an autoclave and treated at 121 ° C. for 40 minutes.
(4) After cooling to 35 ° C., the tubes were taken out, each weighed and diluted with TE buffer so as to be 100 times the initial sample volume. Of these, 1 μL × 2 was used for quantification of template activity.
(5) The template activity was quantified in the same manner as in Example 1.
5 and 6 are amplification curves of samples after the gas phase is sealed with pure nitrogen or air excluding oxygen and subjected to high-temperature and high-pressure treatment, both of which have firm S-shaped curves. Observed, it can be seen that removal of the template activity is inhibited as compared to FIG. 3 of Comparative Example 1 (when sealed with oxygen-containing air). From the above observations, it was confirmed that high-temperature and high-pressure steam treatment in the presence of oxygen is effective for DNA degradation.
［実施例２］
（１）実施例１と同じモデルＰＣＲ廃棄物を使用した。また、限外濾過のスピンカラムを利用してバッファーを蒸留水に置換したサンプルと、ＴＥバッファーに置換したサンプルを調整した。
（２）上記３種類の鋳型ＤＮＡを含む溶液を１０μＬずつ、それぞれ１３本のポリプロピレン製のマイクロチューブ（アシストチューブ型番７２．６９４．１００Ｓ）に分注し、エチレン−プロピレンゴムのＯ−リングを介して専用のネジ口式の蓋で密栓した。また、分注前後のチューブの重量を０.０１μｇの桁で測定し、正確な溶液の分注量を確認した。
（３）チューブを密閉状態のまま、または蓋をせずに完全に気相が飽和の水蒸気と置換する状態でオートクレーブ内に設置し、２０分、４０分、６０分、８０分、１００分、または１２０分間１２１℃で処理した。
（４）３５℃まで冷却後にチューブを取り出し、それぞれ重量を測定して初期サンプル量の１００倍容になるようにＴＥバッファーで希釈した。そのうち１μＬ×２を鋳型活性の定量に用いた。
（５）鋳型活性の定量は実施例１と同様に行った。
図７は蒸留水、ＴＥバッファー、およびＰＣＲ増幅した後のそのままのバッファー組成のサンプルを空気と密閉した状態（■）または蓋をせずに完全に気相が飽和の水蒸気と置換する状態（◆）で一定時間１２１℃で高温高圧処理した後の定量結果で、各処理時間に対するチューブ１本当たりの残存鋳型活性量（コピー数）を表している。鋳型ＤＮＡが含まれる溶液の組成によって分解処理に要する時間に違いはあるが、いずれも空気と密閉して処理したサンプルの方がより早くＤＮＡの鋳型活性を失っている。空気と密閉したものに関しては、長くとも８０分間の処理で鋳型活性の完全除去を達成することができた。
なお、上記実施例１、２では、高温高圧水蒸気処理は１２１℃で処理することで説明したが、１２１℃以上の温度でも同様の効果が期待できる。
[Example 2]
(1) The same model PCR waste as in Example 1 was used. In addition, a sample in which the buffer was replaced with distilled water and a sample in which the TE buffer was replaced were prepared using an ultrafiltration spin column.
(2) 10 μL of the solution containing the above three types of template DNA is dispensed into 13 polypropylene microtubes (assist tube model number 72.694.100S), respectively, through an O-ring of ethylene-propylene rubber. And sealed with a special screw cap lid. In addition, the weight of the tube before and after dispensing was measured with an order of 0.01 μg, and the exact amount of solution dispensed was confirmed.
(3) The tube is placed in an autoclave in a sealed state or in a state where the gas phase is completely replaced with saturated water vapor without a cover, and is placed in an autoclave for 20 minutes, 40 minutes, 60 minutes, 80 minutes, 100 minutes, Or it processed at 121 degreeC for 120 minutes.
(4) After cooling to 35 ° C., the tubes were taken out, each weighed and diluted with TE buffer so as to be 100 times the initial sample volume. Of these, 1 μL × 2 was used for quantification of template activity.
(5) The template activity was quantified in the same manner as in Example 1.
FIG. 7 shows distilled water, TE buffer, and a sample with the same buffer composition after PCR amplification in a state where it is sealed with air (■) or in a state where the vapor phase is completely replaced with water vapor without a lid (◆). ) And the quantitative results after high-temperature and high-pressure treatment at 121 ° C. for a certain time, the residual template activity amount (copy number) per tube for each treatment time is shown. Although there is a difference in the time required for the decomposition treatment depending on the composition of the solution containing the template DNA, in all cases, the sample treated in a sealed manner with air loses the template activity of DNA earlier. For those sealed with air, complete removal of template activity could be achieved with a treatment of at most 80 minutes.
In the first and second embodiments, the high-temperature and high-pressure steam treatment is described as being performed at 121 ° C., but the same effect can be expected even at a temperature of 121 ° C. or higher.
本発明によれば、汚染を出さずにＤＮＡ断片を高効率で分解することが可能であり、近年ＰＣＲ法の普及に伴い問題となっていた、ＰＣＲ鋳型活性を有するＤＮＡ断片を多量に含む医療・実験廃棄物等の処理に有効である。 According to the present invention, a DNA fragment can be decomposed with high efficiency without causing contamination, and has recently become a problem with the spread of the PCR method.・ It is effective for the treatment of experimental waste.
Claims (3)
前記密閉した容器をオートクレーブ内に配置し高温保持することにより、前記密閉した容器内で高温高圧水蒸気処理を行う工程からなる汚染を漏らさずに高効率にＤＮＡを分解除去する方法。 Sealing the aqueous solution containing the DNA fragments in a container with air or a gas containing oxygen;
A method for efficiently decomposing and removing DNA without leaking contamination consisting of a step of performing high-temperature and high-pressure steam treatment in the sealed container by placing the sealed container in an autoclave and maintaining it at a high temperature.
前記密閉した処理装置を排気を行わずに加熱して高温保持することにより、前記密閉した処理装置内で高温高圧水蒸気処理を行う工程からなる汚染を漏らさずに高効率にＤＮＡを分解除去する方法。 A non-sealed container in which wastes containing DNA fragments and waste liquid containing DNA fragments are placed in the processing apparatus, and a step of sealing the processing apparatus with water and with air or a gas containing oxygen;
A method of decomposing and removing DNA with high efficiency without leaking contamination comprising the step of performing high-temperature and high-pressure steam treatment in the sealed processing apparatus by heating the sealed processing apparatus without evacuation and maintaining it at a high temperature. .
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