JP2014097051A - Bacteriolytic reagent for acid-fast bacteria and method of detecting acid-fast bacteria with the same - Google Patents
Bacteriolytic reagent for acid-fast bacteria and method of detecting acid-fast bacteria with the same Download PDFInfo
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Abstract
【課題】 特殊な装置や煩雑な操作を必要とせず、簡便かつ短時間に試料中に含まれる抗酸菌を溶菌して核酸（特にＲＮＡ）を抽出可能な試薬、および前記試薬を用いた試料中に含まれる抗酸菌を検出する方法を提供すること。
【解決手段】 アルキルベンゼンスルホン酸と前記酸との反応により前記酸の金属塩を生成しない中和剤を含む水溶液からなる抗酸菌を溶菌しＲＮＡを抽出するための試薬、前記試薬を抗酸菌を含む試料に添加し加熱処理することで抗酸菌を溶菌しＲＮＡを抽出する方法、および前記方法で得られた抗酸菌由来ＲＮＡを当該ＲＮＡに特異的なプライマー・プローブを用いて増幅・検出することで試料中に含まれる抗酸菌を検出する方法により、前記課題を解決する。
【選択図】 なしPROBLEM TO BE SOLVED: To provide a reagent capable of lysing acid-fast bacteria contained in a sample and extracting nucleic acid (particularly RNA) easily and in a short time without requiring a special apparatus or complicated operation, and a sample using the reagent To provide a method for detecting acid-fast bacteria contained therein.
[MEANS FOR SOLVING PROBLEMS] A reagent for lysing acid-fast bacteria comprising an aqueous solution containing a neutralizing agent that does not produce a metal salt of the acid by reaction of alkylbenzene sulfonic acid and the acid, and extracting the RNA; A method for lysing acid-fast bacteria by extracting the RNA and extracting RNA by heat treatment and adding the sample to the sample, and amplifying the acid-fast bacteria-derived RNA obtained by the above method using a primer / probe specific to the RNA The said subject is solved by the method of detecting the acid-fast bacterium contained in a sample by detecting.
[Selection figure] None
Description
本発明は、抗酸菌を溶菌し核酸を抽出するための試薬、および当該試薬を用いて試料中に含まれる抗酸菌を検出する方法に関する。 The present invention relates to a reagent for lysing acid-fast bacteria and extracting a nucleic acid, and a method for detecting acid-fast bacteria contained in a sample using the reagent.
結核および非結核性抗酸菌症は世界的に蔓延する細菌性疾患であり、当該疾患の根絶が世界的に取り組まれている中、当該疾患であることを迅速に診断することが非常に重要となってきている。また結核と非結核性抗酸菌症とでは感染防護や治療戦略が異なるため、結核菌群と非結核性抗酸菌とを区別して検出することは極めて重要である。 Tuberculosis and non-tuberculous mycobacterial disease are bacterial diseases that are widespread worldwide, and it is very important to quickly diagnose the disease as the disease is being eradicated worldwide. It has become. In addition, since tuberculosis and non-tuberculous mycobacteria have different infection protection and treatment strategies, it is extremely important to distinguish between tuberculosis groups and non-tuberculous mycobacteria.
結核および非結核性抗酸菌（以下あわせて抗酸菌とする）検査の公定法は培養法である。しかしながら抗酸菌（特に結核菌群）の増殖速度は極めて遅く、確定が出るまでに時間を要するため、培養法のみで検査する場合、二次感染の発生や拡大のリスクを有していた。そこで実際の抗酸菌検査業務では、時間のかかる培養法と迅速に抗酸菌の有無を検査可能な方法とを併用して検査している。 The official method for testing tuberculosis and non-tuberculous mycobacteria (hereinafter collectively referred to as acid-fast bacteria) is the culture method. However, the growth rate of mycobacteria (particularly Mycobacterium tuberculosis group) is extremely slow, and it takes time to be confirmed. Therefore, when examining only by the culture method, there is a risk of occurrence or spread of secondary infection. Therefore, in actual acid-fast bacteriological examination work, it is inspected using both a time-consuming culture method and a method capable of quickly examining the presence or absence of acid-fast bacilli.
迅速に抗酸菌の有無を検査可能な方法として、最近注目されている方法は、抗酸菌特異的な核酸（ＤＮＡ／ＲＮＡ）を増幅し検出する方法である。具体的には検査対象の抗酸菌由来の核酸（ＤＮＡ／ＲＮＡ）に特異的なプライマー・プローブを用い、当該核酸を増幅・検出することで、試料中に前記抗酸菌が含まれているか否かを検出する。本方法は、結核菌群由来の核酸に特異的なプライマー・プローブと、非結核性抗酸菌由来の核酸に特異的なプライマー・プローブとを用いることで、結核菌群と非結核性抗酸菌とを区別して検出できるため、迅速な検査方法として好ましい。 As a method capable of rapidly examining the presence or absence of acid-fast bacteria, a method that has recently attracted attention is a method for amplifying and detecting acid-fast bacteria-specific nucleic acid (DNA / RNA). Specifically, by using a primer / probe specific to the nucleic acid (DNA / RNA) derived from the acid-fast bacterium to be tested, whether the acid-fast bacterium is contained in the sample by amplifying and detecting the nucleic acid Detect whether or not. This method uses a primer / probe specific to a nucleic acid derived from a Mycobacterium tuberculosis group and a primer / probe specific to a nucleic acid derived from a non-tuberculous mycobacteria to produce a Mycobacterium tuberculosis group and a nontuberculous antiacid. Since it can be distinguished and detected from bacteria, it is preferable as a rapid inspection method.
抗酸菌特異的な核酸を増幅し検出する方法では、あらかじめ、抗酸菌を溶菌して核酸を抽出する必要がある。抗酸菌を溶菌する方法としては、従来より、有機溶媒等を用いた化学的方法、酵素を用いる生物学的方法、および超音波処理または微粒子存在下での超音波破砕や凍結・融解の繰り返し等の物理的方法等が知られている。また特許文献１には、抗酸菌を含む試料を、非イオン性界面活性剤を含む液体中で加熱処理することで、抗酸菌からＤＮＡを抽出する方法を開示しており、非特許文献１には、有機溶媒や遠心操作を多用することで、抗酸菌からＲＮＡを抽出する方法を開示しており、特許文献２には、抗酸菌を含む試料を界面活性剤を含む液体中で加熱処理後、前記抗酸菌を分離し、当該分離した抗酸菌にジルコニア微粒子を添加して超音波破砕することで、抗酸菌を溶菌しＲＮＡを抽出する方法を開示している。 In the method for amplifying and detecting acid-fast bacterium-specific nucleic acid, it is necessary to lyse the acid-fast bacterium and extract the nucleic acid in advance. Conventional methods of lysing mycobacteria include chemical methods using organic solvents, biological methods using enzymes, and ultrasonic treatment or repeated ultrasonic disruption, freezing and thawing in the presence of fine particles. The physical method etc. are known. Patent Document 1 discloses a method for extracting DNA from acid-fast bacteria by heat-treating a sample containing acid-fast bacteria in a liquid containing a nonionic surfactant. 1 discloses a method of extracting RNA from acid-fast bacteria by using many organic solvents and centrifugal operations. Patent Document 2 discloses a sample containing acid-fast bacteria in a liquid containing a surfactant. After the heat treatment, a method is disclosed in which the acid-fast bacterium is separated, zirconia fine particles are added to the separated acid-fast bacterium, and ultrasonically disrupted to lyse the acid-fast bacterium and extract RNA.
抗酸菌を溶菌して核酸を抽出しようとした場合、抗酸菌は強固な細胞壁を有しているため、処理条件が過酷であったり、処理時間が長くなったり、操作が煩雑であったり、特別な装置を使用する必要があった。またそれに伴いコンタミネーションのリスクもあった。さらに抽出する核酸がＲＮＡの場合、ＤＮＡに比べてアルカリ加水分解に対して脆弱であるため、アルカリ加熱法による簡便な抽出が適用できないという問題もあった。 When an acid-fast bacterium is lysed and nucleic acid is extracted, the acid-fast bacterium has a strong cell wall, so the processing conditions are severe, the processing time is long, and the operation is complicated. There was a need to use special equipment. Along with this, there was a risk of contamination. Further, when the nucleic acid to be extracted is RNA, it is more vulnerable to alkaline hydrolysis than DNA, so that there is a problem that simple extraction by an alkali heating method cannot be applied.
そこで本発明は、特殊な装置や煩雑な操作を必要とせず、簡便かつ短時間に試料中に含まれる抗酸菌を溶菌して核酸（特にＲＮＡ）を抽出可能な試薬、および前記試薬を用いた試料中に含まれる抗酸菌を検出する方法を提供することを目的とする。 Therefore, the present invention uses a reagent that can lyse acid-fast bacteria contained in a sample and extract nucleic acid (particularly RNA) easily and in a short time without requiring a special device or complicated operation, and the reagent. It is an object of the present invention to provide a method for detecting acid-fast bacteria contained in a sample.
本発明者らは、上記目的に鑑みて鋭意検討した結果、本発明を完成するに至った。 As a result of intensive studies in view of the above object, the present inventors have completed the present invention.
すなわち本発明の第一の態様は、アルキルベンゼンスルホン酸と、前記酸との反応により前記酸の金属塩を生成しない中和剤を含む水溶液からなる、抗酸菌を溶菌しＲＮＡを抽出するための試薬である。 That is, the first aspect of the present invention is a method for lysing acid-fast bacteria and extracting RNA, comprising an aqueous solution containing an alkylbenzene sulfonic acid and a neutralizing agent that does not produce a metal salt of the acid by reaction with the acid. It is a reagent.
また本発明の第二の態様は、アルキルベンゼンスルホン酸がドデシルベンゼンスルホン酸である、前記第一の態様に記載の試薬である。 The second aspect of the present invention is the reagent according to the first aspect, wherein the alkylbenzenesulfonic acid is dodecylbenzenesulfonic acid.
また本発明の第三の態様は、中和剤がトリスヒドロキシメチルアミノメタンである、前記第一または第二の態様に記載の試薬である。 A third aspect of the present invention is the reagent according to the first or second aspect, wherein the neutralizing agent is trishydroxymethylaminomethane.
また本発明の第四の態様は、さらに非イオン性界面活性剤を含む、前記第一から第三の態様のいずれかに記載の試薬である。 The fourth aspect of the present invention is the reagent according to any one of the first to third aspects, further comprising a nonionic surfactant.
また本発明の第五の態様は、さらに水溶性有機溶媒を含む、前記第一から第四の態様のいずれかに記載の試薬である。 The fifth aspect of the present invention is the reagent according to any one of the first to fourth aspects, further comprising a water-soluble organic solvent.
さらに本発明の第六の態様は、アルキルベンゼンスルホン酸と、前記酸との反応により前記酸の金属塩を生成しない中和剤を含む水溶液を、抗酸菌を含む試料に添加後、加熱処理することで、抗酸菌を溶菌しＲＮＡを抽出する方法である。 Furthermore, the sixth aspect of the present invention is a method in which an aqueous solution containing an alkylbenzene sulfonic acid and a neutralizing agent that does not produce a metal salt of the acid by the reaction with the acid is added to a sample containing acid-fast bacteria and then heat-treated. This is a method for lysing acid-fast bacteria and extracting RNA.
また本発明の第七の態様は、ドデシルベンゼンスルホン酸とトリスヒドロキシメチルアミノメタンを含む水溶液を、ドデシルベンゼンスルホン酸の終濃度が０．０５％（ｗ／ｖ）以上となるよう、抗酸菌を含む試料に添加後、７０℃から９０℃で５分間から６０分間加熱処理することで、抗酸菌を溶菌しＲＮＡを抽出する方法である。 The seventh aspect of the present invention provides an acid-fast bacterium that contains an aqueous solution containing dodecylbenzenesulfonic acid and trishydroxymethylaminomethane so that the final concentration of dodecylbenzenesulfonic acid is 0.05% (w / v) or more. Is added to a sample containing, and then heat-treated at 70 to 90 ° C. for 5 to 60 minutes to lyse acid-fast bacteria and extract RNA.
さらに本発明の第八の態様は、
アルキルベンゼンスルホン酸と、前記酸との反応により前記酸の金属塩を生成しない中和剤を含む水溶液を、抗酸菌を含む試料に添加後、加熱処理することで、抗酸菌を溶菌しＲＮＡを抽出する工程と、
前記工程で抽出した抗酸菌由来ＲＮＡを当該ＲＮＡに特異的なプライマー・プローブを用いて、増幅・検出する工程とを含む、
試料中に含まれる抗酸菌を検出する方法である。
Furthermore, an eighth aspect of the present invention provides
After adding an aqueous solution containing an alkylbenzene sulfonic acid and a neutralizing agent that does not produce a metal salt of the acid by reaction with the acid to a sample containing acid-fast bacteria, the acid-fast bacteria are lysed by RNA treatment. Extracting the
Amplifying and detecting the mycobacteria-derived RNA extracted in the above step using a primer / probe specific to the RNA,
This is a method for detecting acid-fast bacteria contained in a sample.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明の、抗酸菌を溶菌しＲＮＡを抽出するための試薬（以下、単に本発明の試薬とする）は、アルキルベンゼンスルホン酸と中和剤を少なくとも含むことを特徴としている。 The reagent for lysing acid-fast bacteria and extracting RNA of the present invention (hereinafter simply referred to as the reagent of the present invention) is characterized by containing at least an alkylbenzenesulfonic acid and a neutralizing agent.
本発明の試薬を構成するアルキルベンゼンスルホン酸は、界面活性剤としての機能を有するものであれば特に限定はなく、一例として、デシルベンゼンスルホン酸、ウンデシルベンゼンスルホン酸、ドデシルベンゼンスルホン酸、トリデシルベンゼンスルホン酸、テトラデシルベンゼンスルホン酸といった、炭素数１０から１４の直鎖または分枝鎖を有するアルキル鎖が結合したベンゼンスルホン酸があげられる。中でもドデシルベンゼンスルホン酸（ＤＢＳＡ）は、本発明の試薬を構成するアルキルベンゼンスルホン酸として好ましい態様の一つといえる。 The alkylbenzene sulfonic acid constituting the reagent of the present invention is not particularly limited as long as it has a function as a surfactant. For example, decyl benzene sulfonic acid, undecyl benzene sulfonic acid, dodecyl benzene sulfonic acid, tridecyl Examples thereof include benzenesulfonic acid and tetradecylbenzenesulfonic acid, which are bonded with an alkyl chain having a linear or branched chain having 10 to 14 carbon atoms. Among these, dodecylbenzenesulfonic acid (DBSA) can be said to be one of the preferred embodiments as the alkylbenzenesulfonic acid constituting the reagent of the present invention.
本発明の試薬を構成するアルキルベンゼンスルホン酸はスルホ基（−ＳＯ３Ｈ）を有しているため、水溶液にすると強酸性を示す（一例として、１０％（ｗ／ｖ）ＤＢＳＡ水溶液のｐＨは１以下である）。一方、ＲＮＡは強酸性下では加水分解される可能性がある。そのため、本発明の試薬ではアルキルベンゼンスルホン酸に中和剤を添加することでＲＮＡの加水分解が生じない程度のｐＨに調整する必要がある。ただし本発明の試薬に添加する中和剤として、金属水酸化物（例えば、水酸化ナトリウムや水酸化カリウム）など、アルキルベンゼンスルホン酸との反応により前記酸の金属塩を生成する中和剤を用いるのは好ましくない。その理由として、アルキルベンゼンスルホン酸金属塩は、アルキルベンゼンスルホン酸と比較し溶菌性能が劣るからである（後述の実施例１参照）。本発明の試薬に添加する中和剤の一例として、アンモニアや、２−アミノエタノール、２，２’−イミノジエタノール、２，２’，２’’−ニトリロトリエタノール、２−アミノ−２−メチル−１，３−プロパンジオールなどのアルカノールアミン類や、トリスヒドロキシメチルアミノメタン（Ｔｒｉｓ）があげられる。中でも、毒劇物の扱いを受けず臭気もないＴｒｉｓは、本発明の試薬に添加する中和剤として好ましい態様の一つといえる。 Since the alkylbenzene sulfonic acid constituting the reagent of the present invention has a sulfo group (—SO 3 H), it exhibits strong acidity in an aqueous solution (for example, the pH of a 10% (w / v) DBSA aqueous solution is 1 Below). On the other hand, RNA may be hydrolyzed under strong acidity. Therefore, in the reagent of the present invention, it is necessary to adjust the pH to such an extent that RNA is not hydrolyzed by adding a neutralizing agent to alkylbenzenesulfonic acid. However, as a neutralizing agent to be added to the reagent of the present invention, a neutralizing agent that generates a metal salt of the acid by reaction with an alkylbenzene sulfonic acid such as a metal hydroxide (for example, sodium hydroxide or potassium hydroxide) is used. Is not preferred. This is because the alkylbenzenesulfonic acid metal salt is inferior in lysis performance as compared to alkylbenzenesulfonic acid (see Example 1 described later). Examples of the neutralizing agent added to the reagent of the present invention include ammonia, 2-aminoethanol, 2,2′-iminodiethanol, 2,2 ′, 2 ″ -nitrilotriethanol, 2-amino-2-methyl- Examples include alkanolamines such as 1,3-propanediol and trishydroxymethylaminomethane (Tris). Among these, Tris that is not treated with poisonous and deleterious substances and has no odor can be said to be one of preferred embodiments as a neutralizing agent added to the reagent of the present invention.
本発明の試薬におけるアルキルベンゼンスルホン酸の濃度は、アルキルベンゼンスルホン酸がＤＢＳＡの場合、抗酸菌を含む試料に本発明の試薬を添加した時点の終濃度で０．０５％（ｗ／ｖ）以上であればよく、０．５％（ｗ／ｖ）以上であるとより好ましい。上限については特に限定はないが、アルキルベンゼンスルホン酸がＤＢＳＡの場合、２０％（ｗ／ｖ）以上の水溶液は粘性が高く、取り扱いが困難となるため、避けたほうが好ましい。なおアルキルベンゼンスルホン酸は、粘性が高く、水への溶解速度も遅い。そのため本発明の試薬を調製する際、あらかじめアルキルベンゼンスルホン酸に、２−プロパノールといった低級アルコールや、エチレングリコールといったジオールや、ジメチルスルフォキシドといった非プロトン性有機溶媒などの水溶性の有機溶媒を混和させてから調製すると、粘性を下げることができ、かつ水への溶解速度も向上するため好ましい。 When the alkylbenzene sulfonic acid is DBSA, the concentration of the alkylbenzene sulfonic acid in the reagent of the present invention is 0.05% (w / v) or more at the final concentration when the reagent of the present invention is added to the sample containing acid-fast bacteria. What is necessary is just 0.5% (w / v) or more. The upper limit is not particularly limited, but when the alkylbenzenesulfonic acid is DBSA, an aqueous solution of 20% (w / v) or more is preferably avoided because it has high viscosity and is difficult to handle. Alkylbenzenesulfonic acid has a high viscosity and a low dissolution rate in water. Therefore, when preparing the reagent of the present invention, a water-soluble organic solvent such as a lower alcohol such as 2-propanol, a diol such as ethylene glycol, or an aprotic organic solvent such as dimethyl sulfoxide is mixed with the alkylbenzene sulfonic acid in advance. It is preferable to prepare it afterwards because the viscosity can be lowered and the dissolution rate in water is improved.
本発明の試薬の好ましい態様として、非イオン性界面活性剤をさらに添加した試薬があげられ、前記試薬により溶菌効率のばらつきを改善することができる。添加する非イオン性界面活性剤は、当業者が通常用いるものから適宜選択することができ、その一例として、ＭＥＧＡ−８（ｎ−Ｏｃｔａｎｏｙｌ−Ｎ−ｍｅｔｈｙｌ−Ｄ−ｇｌｕｃａｍｉｎｅ）、ＭＥＧＡ−９（ｎ−Ｎｏｎａｎｏｙｌ−Ｎ−ｍｅｔｈｙｌ−Ｄ−ｇｌｕｃａｍｉｎｅ）、ＭＥＧＡ−１０（ｎ−Ｄｅｃａｎｏｙｌ−Ｎ−ｍｅｔｈｙｌ−Ｄ−ｇｌｕｃａｍｉｎｅ）、ｎ−ドデシル−β−Ｄ−マルトシド、オクチル−β−Ｄ−グルコシド、Ｔｒｉｔｏｎ Ｘ−１００（商品名）があげられる。濃度については、臨界ミセル濃度（ＣＭＣ）を考慮のうえ、適宜設定すればよい。 A preferred embodiment of the reagent of the present invention is a reagent to which a nonionic surfactant is further added, and the variation in lysis efficiency can be improved by the reagent. The nonionic surfactant to be added can be appropriately selected from those usually used by those skilled in the art. Examples thereof include MEGA-8 (n-octanoyl-N-methyl-D-glucamine), MEGA-9 (n -Nonanoyl-N-methyl-D-glucomine), MEGA-10 (n-Decanyl-N-methyl-D-glucomine), n-dodecyl-β-D-maltoside, octyl-β-D-glucoside, Triton X- 100 (trade name). The concentration may be appropriately set in consideration of the critical micelle concentration (CMC).
本発明の試薬を抗酸菌を含む試料に添加し、加熱処理することで、抗酸菌を溶菌しＲＮＡを抽出することができる。加熱処理する温度は６０℃以下では抗酸菌が実質的に溶菌しないことから（Ｃｈｅｍｉｃａｌ Ｅｎｇｉｎｅｅｒｉｎｇ Ｓｃｉｅｎｃｅ，６４（９），１９４４−１９５１（２００９））、６５℃から９５℃までの範囲内であればよく、７０℃から９０℃までの範囲内が特に好ましい。加熱処理する時間は温度に依存するが、加熱処理する温度が７０℃から９０℃までの場合、５分から６０分までの間とすればよく、１０分前後が特に好ましい。 By adding the reagent of the present invention to a sample containing acid-fast bacteria and heat treatment, the acid-fast bacteria can be lysed and RNA can be extracted. Since the acid-fast bacteria are not substantially lysed at a temperature of 60 ° C. or less (Chemical Engineering Science, 64 (9), 1944-951 (2009)), the heat treatment temperature is within the range from 65 ° C. to 95 ° C. In particular, the range from 70 ° C. to 90 ° C. is particularly preferable. The time for the heat treatment depends on the temperature, but when the temperature for the heat treatment is from 70 ° C. to 90 ° C., it may be from 5 minutes to 60 minutes, and about 10 minutes is particularly preferable.
本発明の試薬を抗酸菌を含む試料に添加し、加熱処理することで、抗酸菌を溶菌して抗酸菌由来ＲＮＡを抽出した後は、当該ＲＮＡに特異的なプライマー・プローブを用いて、増幅・検出することにより、試料中に含まれる抗酸菌を検出することができる。なおＲＮＡを抽出した後、ＲＮｅａｓｙ（ＱＩＡＧＥＮ）などの市販のＲＮＡ精製キットを用いてＲＮＡを高純度に精製してから、前記増幅・検出を行なってもよい。また前記増幅・検出は、ＴＭＡ（Ｔｒａｎｓｃｒｉｐｔｉｏｎ−Ｍｅｄｉａｔｅｄ Ａｍｐｌｉｆｉｃａｔｉｏｎ）法、ＮＡＳＢＡ（Ｎｕｃｌｅｉｃ Ａｃｉｄ Ｓｅｑｕｅｎｃｅ−Ｂａｓｅｄ Ａｍｐｌｉｆｉｃａｔｉｏｎ）法、ＴＲＣ（Ｔｒａｎｓｃｒｉｐｔｉｏｎ Ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ Ｃｏｎｃｅｒｔｅｄ ｒｅａｃｔｉｏｎ）（特開２０００−０１４４００号公報）法など従来より知られているＲＮＡ増幅法を用いて行なえばよい。 After adding the reagent of the present invention to a sample containing acid-fast bacteria and heat-treating it, the acid-fast bacteria are lysed and the acid-fast bacteria-derived RNA is extracted, and then a primer / probe specific to the RNA is used. Thus, acid-fast bacteria contained in the sample can be detected by amplification and detection. In addition, after extracting RNA, you may perform said amplification and detection, after purifying RNA with high purity using commercially available RNA purification kits, such as RNeasy (QIAGEN). In addition, the amplification and detection are performed by the TMA (Transcribation-Mediated Amplification) method, the NASBA (Nucleic Acid Sequence-Based Amplification) method, the TRC (Transcribion Reverse-Transcribion Method 400), and the like. The RNA amplification method may be used.
本発明の試薬で溶菌可能な抗酸菌は、Ｍｙｃｏｂａｃｔｅｒｉｕｍ属に属する菌のことを指し、具体的には、Ｍ．ａｖｉｕｍ、Ｍ．ｉｎｔｒａｃｅｌｌｕｌａｒａｅ、Ｍ．ｇｏｒｄｏｎａｅ、Ｍ．ｔｕｂｅｒｃｕｌｏｓｉｓ（結核菌）、Ｍ．ｋａｎｓａｓｉｉ、Ｍ．ｆｏｒｔｕｉｔｕｍ、Ｍ．ｃｈｅｌｏｎａｅ、Ｍ．ｂｏｖｉｓ、Ｍ．ｓｃｒｏｆｕｌａｃｅｕｍ、Ｍ．ｐａｒａｔｕｂｅｒｃｕｌｏｓｉｓ（ヨーネ菌）、Ｍ．ｐｈｌｅｉ、Ｍ．ｍａｒｉｎｕｍ、Ｍ．ｓｉｍｉａｅ、Ｍ．ｓｚｕｌｇａｉ、Ｍ．ｌｅｐｒａｅ（らい菌）、Ｍ．ｘｅｎｏｐｉ、Ｍ．ｕｌｃｅｒａｎｓ、Ｍ．ｌｅｐｒａｅｍｕｒｉｕｍ、Ｍ．ｆｌａｖｅｓｃｅｎｓ、Ｍ．ｔｅｒｒａｅ、Ｍ．ｎｏｎｃｈｒｏｍｏｇｅｎｉｃｕｍ、Ｍ．ｍａｌｍｏｅｎｓｅ、Ｍ．ａｓｉａｔｉｃｕｍ、Ｍ．ｖａｃｃａｅ、Ｍ．ｇａｓｔｒｉ、Ｍ．ｔｒｉｖｉａｌｅ、Ｍ．ａｆｒｉｃａｎｕｍ、Ｍ．ｔｈｅｒｍｏｒｅｓｉｓｔａｂｌｅ、Ｍ．ｓｍｅｇｍａｔｉｓ、Ｍ．ｓｈｉｎｊｕｋｕｅｎｓｅが例示できる。試料中に含まれる抗酸菌を検出するには、前述した抗酸菌のＲＮＡに特異的なプライマー・プローブを設計し、当該設計したプライマー・プローブを用いて適切な条件下でＲＮＡの増幅・検出を行なえばよい（例えば、特開２００４−１９４５８３号公報）。 The acid-fast bacterium that can be lysed by the reagent of the present invention refers to a bacterium belonging to the genus Mycobacterium, specifically, avium, M.M. intracellularae, M .; gordonae, M.M. tuberculosis, M. tuberculosis. Kansasii, M.M. fortuitum, M.M. chelonae, M.C. bovis, M.M. s. paratuberculosis, M. pneumoniae. phlei, M.M. marinum, M.M. simiae, M.M. szulgai, M.M. leprae, M. et al. xenopi, M.M. ulcerans, M.M. lepraemurium, M. et al. flavescens, M.M. terrae, M.M. nonchromogenicum, M.M. malmoense, M.M. asiaticum, M. et al. vaccae, M.M. gastri, M.M. triviale, M.M. africanum, M.M. thermoresistable, M.M. smegmatis, M.M. Shinjukuense can be exemplified. To detect acid-fast bacilli contained in a sample, a primer / probe specific to the acid-fast bacilli RNA described above is designed, and RNA amplification / ampling under appropriate conditions using the designed primer / probe is performed. What is necessary is just to perform a detection (for example, Unexamined-Japanese-Patent No. 2004-194583).
本発明における抗酸菌を含む試料の一例として、喀痰、気管支洗浄液、気管支肺胞洗浄液、胃液、血液、骨髄液、尿、糞便、組織、その他体液、培養液などがあげられる。なお、抗酸菌を含む試料が喀痰の場合は、ＮＡＬＣ（Ｎ−ａｃｅｔｙｌ−Ｌ−ｃｙｓｔｅｉｎｅ）−ＮａＯＨ法などの公知の方法によりあらかじめ試料の粘性を下げる前処理を行なったほうがよい。 Examples of samples containing acid-fast bacteria in the present invention include sputum, bronchial lavage fluid, bronchoalveolar lavage fluid, gastric fluid, blood, bone marrow fluid, urine, feces, tissue, other body fluids, culture fluid, and the like. In addition, when the sample containing an acid-fast bacterium is sputum, it is better to perform a pretreatment for reducing the viscosity of the sample in advance by a known method such as a NALC (N-acetyl-L-cysteine) -NaOH method.
本発明の試薬は、アルキルベンゼンスルホン酸と、前記酸との反応により前記酸の金属塩を生成しない中和剤を少なくとも含むことを特徴としている。抗酸菌を含む試料に本発明の試薬を添加し加熱処理することで抗酸菌を溶菌しＲＮＡを抽出後、当該ＲＮＡに特異的なプライマー・プローブを用いて増幅・検出することで、試料中に含まれる抗酸菌を簡便かつ短時間に検査することができる。また本発明の試薬を用いた抗酸菌の溶菌操作は、１本のチューブで完結し、ヒートブロック以外の特別な装置が不要なため、容易に実施することができる。またコンタミネーションリスクが少なく、バイオセーフティも確保できる操作である。 The reagent of the present invention is characterized in that it contains at least a neutralizing agent that does not produce a metal salt of the acid by a reaction between the alkylbenzenesulfonic acid and the acid. By adding the reagent of the present invention to a sample containing acid-fast bacteria and heat-treating it, the acid-fast bacterium is lysed and RNA is extracted, and then amplified and detected using a primer / probe specific to the RNA. The acid-fast bacterium contained therein can be inspected easily and in a short time. Further, the lysis operation of acid-fast bacteria using the reagent of the present invention can be easily carried out because it is completed with one tube and no special apparatus other than the heat block is required. In addition, it is an operation that has little risk of contamination and can ensure biosafety.
以下、本発明をさらに詳細に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described in more detail, but the present invention is not limited thereto.
実施例１ 界面活性剤による抗酸菌から溶菌効率の検討
各種界面活性剤により抗酸菌の溶菌操作を行ない、溶菌効率を、前記操作により液相に放出された抗酸菌由来のＲＮＡを増幅・検出することで評価した。
（１）ＭｙｃｏＢｒｏｔｈ（極東製薬）中で静置培養したＭｙｃｏｂａｃｔｅｒｉｕｍ ｂｏｖｉｓ ＢＣＧ Ｔｏｋｙｏ株（以下、ＢＣＧと記載）を孔径５μｍの親水性シリンジフィルター（日本ＰＡＬＬ）でろ過後、ろ液の６００ｎｍにおける吸光度（ＯＤ６００）を測定し、その吸光度を基にＯＤ６００が０．１になるように滅菌蒸留水で希釈した（１０７ｃｆｕ（ｃｏｌｏｎｙ ｆｏｒｍｉｎｇ ｕｎｉｔ）／ｍＬ相当）。
（２）（１）の希釈液を段階希釈し、１０２ｃｆｕのＢＣＧを含む２００μＬの菌液を調製した。
（３）（２）で調製した菌液２００μＬに、１０％（ｗ／ｖ）ドデシルベンゼンスルホン酸（ＤＢＳＡ）水溶液、１０％（ｗ／ｖ）ドデシルベンゼンスルホン酸ナトリウム塩（Ｎａ−ＤＢＳ）水溶液、１０％（ｗ／ｖ）ドデシル硫酸ナトリウム（ＳＤＳ）水溶液、または１０％（ｗ／ｖ）セチルトリメチルアンモニウムブロミド（ＣＴＡＢ）水溶液、各２００μＬを添加し混和後、８０℃で１０分加熱処理することで溶菌操作を行なった。なお１０％（ｗ／ｖ）ＤＢＳＡ水溶液はｐＨ１以下の強酸性水溶液であり、ＲＮＡが加水分解される可能性があるので、あらかじめトリスヒドロキシメチルアミノメタン（Ｔｒｉｓ）で中和している。
（４）（３）の処理液のうち２００μＬを、ＲＮｅａｓｙ ｍｉｎｉ ｋｉｔ（ＱＩＡＧＥＮ）を用いることで精製操作を行ない、溶出液５０μＬを得た。なお本操作は、キットの取扱説明書に従い実施した。
（５）（４）の溶出液５μＬを、市販の結核菌群ｒＲＮＡ検出試薬（ＴＲＣＲａｐｉｄ Ｍ．ＴＢ、東ソー）およびＴＲＣＲリアルタイムモニター（ＴＲＣＲａｐｉｄ−１６０、東ソー）を用いて溶出液中のＲＮＡを増幅検出し、前記モニターで陽性判定となる時間（陽性時間）を測定した。なお本操作は、前記試薬の添付文書および前記モニターの取扱説明書に従い実施した。陽性時間が短いほど測定試料中のＢＣＧ ＲＮＡ量が多いことから、溶菌効率がよいといえる。
Example 1 Examination of lysis efficiency from acid-fast bacteria with surfactants Lysis of acid-fast bacteria was performed with various surfactants, and the lysis efficiency was amplified by amplification of RNA derived from acid-fast bacteria released into the liquid phase by the above-described operation.・ Evaluated by detecting.
(1) Mycobacterium bovis BCG Tokyo strain (hereinafter referred to as BCG) statically cultured in MycoBroth (Kyokuto Pharmaceutical) was filtered with a hydrophilic syringe filter (Japan PALL) having a pore size of 5 μm, and the absorbance at 600 nm of the filtrate (OD600) ) Was measured and diluted with sterilized distilled water based on the absorbance so that the OD600 was 0.1 (equivalent to 10 7 cfu (colony forming unit) / mL).
(2) The diluted solution of (1) was serially diluted to prepare 200 μL of a bacterial solution containing 10 2 cfu of BCG.
(3) 10% (w / v) dodecylbenzenesulfonic acid (DBSA) aqueous solution, 10% (w / v) sodium dodecylbenzenesulfonic acid sodium salt (Na-DBS) aqueous solution, to 200 μL of the bacterial solution prepared in (2), By adding 200 μL each of 10% (w / v) sodium dodecyl sulfate (SDS) aqueous solution or 10% (w / v) cetyltrimethylammonium bromide (CTAB) aqueous solution and mixing, heat treatment is performed at 80 ° C. for 10 minutes. Lysis operation was performed. The 10% (w / v) DBSA aqueous solution is a strongly acidic aqueous solution having a pH of 1 or less, and RNA may be hydrolyzed, so it is neutralized with trishydroxymethylaminomethane (Tris) in advance.
(4) 200 μL of the processing solution of (3) was purified using RNeasy mini kit (QIAGEN) to obtain 50 μL of eluate. This operation was performed according to the instruction manual of the kit.
(5) 5 μL of the eluate of (4) was amplified and detected using a commercially available tuberculosis group rRNA detection reagent (TRCRapid M.TB, Tosoh) and a TRCR real-time monitor (TRCRapid-160, Tosoh). Then, the time (positive time) during which a positive determination was made on the monitor was measured. This operation was performed according to the attached document of the reagent and the instruction manual of the monitor. Since the amount of BCG RNA in the measurement sample is larger as the positive time is shorter, it can be said that the lysis efficiency is better.
溶菌操作（前記（３）の操作）は３重で行ない、精製操作と測定（前記（４）および（５））は各処理液に対し１重で行なった。結果を表１に示す。なお表１中、Ｎ．Ｄ．は標的ＲＮＡを検出できなかったことを表す。 The lysis operation (operation (3) above) was performed in triplicate, and the purification operation and measurement (above (4) and (5)) were performed in a single layer for each treatment solution. The results are shown in Table 1. In Table 1, N.I. D. Indicates that the target RNA could not be detected.
実施例２ ＤＢＳＡの最適濃度検討
実施例１で検討した界面活性剤のうち、抗酸菌を最も良好に溶菌したＤＢＳＡについて最適濃度を検討した。
（１）実施例１（１）の希釈液から滅菌蒸留水で段階希釈し、１０２ｃｆｕのＢＣＧを含む１００μＬの菌液を調製した。
（２）あらかじめＴｒｉｓで中和した１０％（ｗ／ｖ）ＤＢＳＡ水溶液を蒸留水で段階希釈し、１％（ｗ／ｖ）、０．１％（ｗ／ｖ）および０．０１％（ｗ／ｖ）ＤＢＳＡ−Ｔｒｉｓ水溶液を調製した。
（３）（１）で調製した菌液１００μＬに、（２）で調製したＤＢＳＡ−Ｔｒｉｓ水溶液１００μＬを添加し混和後、８０℃で１０分加熱処理することで溶菌操作を行なった。なお、１０％（ｗ／ｖ）ＤＢＳＡ−Ｔｒｉｓ水溶液を添加した場合の終濃度は５％（ｗ／ｖ）、１％（ｗ／ｖ）ＤＢＳＡ−Ｔｒｉｓ水溶液を添加した場合の終濃度は０．５％（ｗ／ｖ）、０．１％（ｗ／ｖ）ＤＢＳＡ−Ｔｒｉｓ水溶液を添加した場合の終濃度は０．０５％（ｗ／ｖ）、０．０１％（ｗ／ｖ）ＤＢＳＡ−Ｔｒｉｓ水溶液を添加した場合の終濃度は０．００５％（ｗ／ｖ）となる。
（４）（３）の処理液全量（２００μＬ）を実施例１（４）と同様な方法で精製し、実施例１（５）と同様な方法で陽性時間を測定した。
Example 2 Examination of optimum concentration of DBSA Among the surfactants examined in Example 1, the optimum concentration was examined for DBSA that best lyses acid-fast bacteria.
(1) From the diluted solution of Example 1 (1), serially diluted with sterilized distilled water to prepare 100 μL of a bacterial solution containing 10 2 cfu of BCG.
(2) A 10% (w / v) DBSA aqueous solution previously neutralized with Tris was serially diluted with distilled water, and 1% (w / v), 0.1% (w / v) and 0.01% (w / V) A DBSA-Tris aqueous solution was prepared.
(3) To 100 μL of the bacterial solution prepared in (1), 100 μL of the DBSA-Tris aqueous solution prepared in (2) was added and mixed, followed by heat treatment at 80 ° C. for 10 minutes to perform the lysis operation. The final concentration when the 10% (w / v) DBSA-Tris aqueous solution was added was 5% (w / v), and the final concentration when the 1% (w / v) DBSA-Tris aqueous solution was added was 0.00. When 5% (w / v) and 0.1% (w / v) DBSA-Tris aqueous solution is added, final concentrations are 0.05% (w / v) and 0.01% (w / v) DBSA- When the Tris aqueous solution is added, the final concentration is 0.005% (w / v).
(4) The total amount (200 μL) of the processing solution of (3) was purified by the same method as in Example 1 (4), and the positive time was measured by the same method as in Example 1 (5).
結果を表２に示す。なお表２中、Ｎ．Ｄ．は標的ＲＮＡを検出できなかったことを表す。 The results are shown in Table 2. In Table 2, N. D. Indicates that the target RNA could not be detected.
実施例３ 非イオン性界面活性剤の添加の効果（その１）
ＤＢＳＡに非イオン界面活性剤をさらに添加することで、抗酸菌の溶菌効率がさらに向上するか評価した。
Example 3 Effect of Addition of Nonionic Surfactant (Part 1)
It was evaluated whether the lysis efficiency of acid-fast bacteria was further improved by further adding a nonionic surfactant to DBSA.
実施例１（２）で調製した菌液２００μＬに添加する水溶液として、２．８ｍｇ／ｍＬのＭＥＧＡ−１０（ｎ−Ｄｅｃａｎｏｙｌ−Ｎ−ｍｅｔｈｙｌ−Ｄ−ｇｌｕｃａｍｉｎｅ）（同仁化学）を含む１０％（ｗ／ｖ）ＤＢＳＡ−Ｔｒｉｓ水溶液２００μＬとした他は、実施例１と同様な方法で評価した。結果を表３に示す。 10% (w) containing 2.8 mg / mL MEGA-10 (n-Decanyl-N-methyl-D-glucomine) (Dojindo) as an aqueous solution to be added to 200 μL of the bacterial solution prepared in Example 1 (2) / V) Evaluation was performed in the same manner as in Example 1 except that 200 μL of the DBSA-Tris aqueous solution was used. The results are shown in Table 3.
実施例４ 非イオン性界面活性剤の添加の効果（その２）
ＤＢＳＡにＭＥＧＡ−１０以外の非イオン界面活性剤を添加することで、ＭＥＧＡ−１０を添加したときと同様な効果が得られるか検討した。
Example 4 Effect of Addition of Nonionic Surfactant (Part 2)
It was examined whether the same effect as when MEGA-10 was added could be obtained by adding a nonionic surfactant other than MEGA-10 to DBSA.
実施例１（２）で調製した菌液２００μＬに添加する水溶液として、以下に示す（Ａ）から（Ｆ）の水溶液２００μＬとした他は、実施例１と同様な方法で評価した。
（Ａ）２．８ｍｇ／ｍＬのＭＥＧＡ−８（ｎ−Ｏｃｔａｎｏｙｌ−Ｎ−ｍｅｔｈｙｌ−Ｄ−ｇｌｕｃａｍｉｎｅ）（同仁化学）を含む１０％（ｗ／ｖ）ＤＢＳＡ−Ｔｒｉｓ水溶液
（Ｂ）２．８ｍｇ／ｍＬのＭＥＧＡ−９（ｎ−Ｎｏｎａｎｏｙｌ−Ｎ−ｍｅｔｈｙｌ−Ｄ−ｇｌｕｃａｍｉｎｅ）（同仁化学）を含む１０％（ｗ／ｖ）ＤＢＳＡ−Ｔｒｉｓ水溶液
（Ｃ）２．８ｍｇ／ｍＬのＭＥＧＡ−１０（同仁化学）を含む１０％（ｗ／ｖ）ＤＢＳＡ−Ｔｒｉｓ水溶液
（Ｄ）２．８ｍｇ／ｍＬのｎ−ドデシル−β−Ｄ−マルトシド（１２ｍ）を含む１０％（ｗ／ｖ）ＤＢＳＡ−Ｔｒｉｓ水溶液
（Ｅ）２．８ｍｇ／ｍＬのｎ−オクチル−β−Ｄ−グルコシド（８ｇ）を含む１０％（ｗ／ｖ）ＤＢＳＡ−Ｔｒｉｓ水溶液
（Ｆ）２．８ｍｇ／ｍＬのＴｒｉｔｏｎ Ｘ−１００（ＴＸ）（商品名）を含む１０％（ｗ／ｖ）ＤＢＳＡ−Ｔｒｉｓ水溶液
結果を表４に示す。本実施例で検討した５種類の非イオン性界面活性剤のいずれもがＭＥＧＡ−１０と同様、溶菌効率のばらつきが改善されていることがわかる。
Evaluation was performed in the same manner as in Example 1 except that 200 μL of the aqueous solutions (A) to (F) shown below were used as the aqueous solution to be added to 200 μL of the bacterial solution prepared in Example 1 (2).
(A) 10% (w / v) DBSA-Tris aqueous solution (B) containing 2.8 mg / mL MEGA-8 (n-Octanoyl-N-methyl-D-glucamine) (Dojin Chemical) 2.8 mg / mL 10% (w / v) DBSA-Tris aqueous solution (C) containing MEGA-9 (n-Nonanoyl-N-methyl-D-glucamine) (Dojindo) 2.8 mg / mL MEGA-10 (Dojindo) 10% (w / v) DBSA-Tris aqueous solution (D) containing 10% (w / v) DBSA-Tris aqueous solution (E) containing 2.8 mg / mL n-dodecyl-β-D-maltoside (12 m) 10% (w / v) DBSA-Tris aqueous solution (F) containing 2.8 mg / mL n-octyl-β-D-glucoside (8 g) 2.8 mg / mL Tri Table 4 shows the results of 10% (w / v) DBSA-Tris aqueous solution containing ton X-100 (TX) (trade name). It can be seen that all of the five types of nonionic surfactants studied in this example have improved lysis efficiency variations as in MEGA-10.
ＤＢＳＡとＭＥＧＡ−１０とを含む水溶液で抗酸菌を溶菌する際の加熱処理温度および加熱処理時間の検討を行なった。
（１）実施例１（２）で調製した菌液２００μＬに、２．８ｍｇ／ｍＬのＭＥＧＡ−１０（同仁化学）と１０％（ｗ／ｖ）ＤＢＳＡとを含む水（９０％）／２−プロパノール（１０％）混合溶液（Ｔｒｉｓで中和）２００μＬを添加し、混和した。なお２−プロパノールは、ＤＢＳＡの粘性の低下と水への溶解性の向上を目的に添加している。
（２）７０℃、８０℃または９０℃で５分間、１０分間、３０分間または６０分間加熱処理後、前記処理液１００μＬを実施例１（４）と同様な方法で精製し、実施例１（５）と同様な方法で陽性時間を測定した。
(1) Water (90%) / 2− containing 200 μL of the bacterial solution prepared in Example 1 (2) and containing 2.8 mg / mL MEGA-10 (Dojindo) and 10% (w / v) DBSA 200 μL of propanol (10%) mixed solution (neutralized with Tris) was added and mixed. 2-Propanol is added for the purpose of reducing the viscosity of DBSA and improving the solubility in water.
(2) After heat treatment at 70 ° C., 80 ° C. or 90 ° C. for 5 minutes, 10 minutes, 30 minutes or 60 minutes, 100 μL of the treatment solution was purified by the same method as in Example 1 (4), and Example 1 ( The positive time was measured by the same method as in 5).
結果を表５に示す。本実施例で検討した加熱処理温度（７０℃から９０℃）および加熱処理時間（５分から６０分）の範囲であれば、溶菌効率に大きな差はないことがわかる。また本実施例では、ＤＢＳＡの粘性の低下と水への溶解性の向上を目的に２−プロパノールを添加したが、溶菌自体には大きな影響を与えていないことがわかる。 The results are shown in Table 5. It can be seen that there is no significant difference in lysis efficiency within the range of the heat treatment temperature (70 ° C. to 90 ° C.) and the heat treatment time (5 minutes to 60 minutes) studied in this example. In this example, 2-propanol was added for the purpose of lowering the viscosity of DBSA and improving the solubility in water, but it was found that the lysis itself was not greatly affected.
実施例５では、ＤＢＳＡの粘性の低下と水への溶解性の向上を目的に２−プロパノールを添加したが、他の水溶性有機溶媒も同様に適用可能か検討した。
２−プロパノール（２−ＰｒＯＨ）の代わりに、非プロトン性有機溶媒であるジメチルスルフォキシド（ＤＭＳＯ）またはジオールであるエチレングリコール（Ｅｔ−Ｇｌｙ）を用い、加熱処理温度・加熱処理時間を８０℃・１０分とした他は、実施例５と同様な方法で実施した。結果を表６に示す。本実施例で検討した水溶性有機溶媒はいずれも使用可能であることがわかる。 Instead of 2-propanol (2-PrOH), dimethyl sulfoxide (DMSO), which is an aprotic organic solvent, or ethylene glycol (Et-Gly), which is a diol, is used. -It implemented by the method similar to Example 5 except having set it as 10 minutes. The results are shown in Table 6. It can be seen that any of the water-soluble organic solvents studied in this example can be used.
結核菌を含む抗酸菌の検査では、操作者のバイオセーフティが確保されていることが重要である。抗酸菌検査における塗抹検査の陽性最高値は３＋であり、これは１０６ｃｆｕ（ｃｏｌｏｎｙ ｆｏｒｍｉｎｇ ｕｎｉｔ）／ｍＬに相当する（結核 第８３巻 第４号 ３８７−３９０、２００８年）。また、液体培養の陽性時の菌数は１０６から１０７ｃｆｕ／ｍＬである（日本ＢＤ 培養同定・抗酸菌キット「ミジット分離培養剤」 添付文書）。そこで本発明者らは、検体が濃厚試料または培養液の場合は、１０７ｃｆｕ程度の抗酸菌が検体に持ち込まれ得ると考え、１０７ｃｆｕの抗酸菌を本発明の試薬で処理した液から生菌が検出されない、すなわちバイオセーフティが確保されているかどうかを検討した。
（１）ＭｙｃｏＢｒｏｔｈ（極東製薬）中で静置培養したＢＣＧを孔径５μｍの親水性シリンジフィルター（日本ＰＡＬＬ）でろ過後、ろ液の６００ｎｍにおける吸光度（ＯＤ６００）を測定し、その吸光度を基にＯＤ６００が０．１になるように、氷冷した滅菌蒸留水で希釈した（１０７ｃｆｕ／ｍＬ相当）。
（２）希釈した菌液を１ｍＬ分取後、１６０００Ｇで１０分間、４℃の条件で遠心（以下、前記条件を「強遠心」と記載）し、上清を慎重に取り除いた後、沈殿を２００μＬの滅菌蒸留水に懸濁させた。得られたＢＣＧ懸濁液を抗酸菌を含む試料として、以下に示す溶菌処理を行なった。
（３）（２）で調製した菌液２００μＬに対し、１２％（ｗ／ｖ）の２−プロパノール、と２．８ｍｇ／ｍＬのＭＥＧＡ−１０とを含む１０％（ｗ／ｖ）ＤＢＳＡ水溶液（Ｔｒｉｓで中和）、または滅菌蒸留水を、それぞれ２００μＬ添加し、混和した。
（４）（３）で混和した液のうち、ＤＢＳＡ水溶液と混和した液は７分間、９分間、１０分間または１５分間、蒸留水と混和した液は１０分間、それぞれ７０℃または８０℃で加熱した。加熱後は氷上に５分以上静置した。前記処理は、それぞれの温度・時間で２重で行なった。
（５）加熱処理後、強遠心により上清を取り除いた後、沈殿に０．０５％（ｗ／ｖ）のＴｗｅｅｎ ８０（Ｓｉｇｍａ）を含む１／１５Ｍのリン酸緩衝液（以下、Ｔ−ＰＢと記載)を１ｍＬ加え、よく撹拌することで沈殿を洗浄した。
（６）（５）の洗浄を再度行なった後、最終的に沈殿を３００μＬのＴ−ＰＢに懸濁させ、全量を７Ｈ９−Ｃ平板培地（極東製薬）に塗布し、３７℃、５％ＣＯ２の条件で培養した。培養期間は結核菌検査指針２００７（日本結核病学会抗酸菌検査法検討委員会編）に従って８週間とした。なお（１）で希釈した菌液が正しく１０７ｃｆｕ／ｍＬに調製されたかどうかを確認する目的で、当該菌液から、Ｔ−ＰＢを用いて１０５倍に段階希釈した液を７Ｈ９−Ｃ平板培地に塗布したものを培養陽性として同時に培養した。
（７）コロニーが認められた時点でコロニー数をカウントした。８週間後でもコロニーが認められないものはＮ．Ｄ．とした。
(1) After filtering BCG statically cultured in MycoBroth (Kyokuto Pharmaceutical Co., Ltd.) with a hydrophilic syringe filter (Japan PALL) having a pore size of 5 μm, the absorbance at 600 nm (OD600) of the filtrate is measured, and OD600 based on the absorbance. Was diluted with ice-cooled sterilized distilled water so as to be 0.1 (equivalent to 10 7 cfu / mL).
(2) After 1 mL of the diluted bacterial solution is collected, it is centrifuged at 16000 G for 10 minutes at 4 ° C. (hereinafter referred to as “strong centrifugation”), the supernatant is carefully removed, and the precipitate is removed. Suspended in 200 μL of sterile distilled water. The obtained BCG suspension was used as a sample containing acid-fast bacteria, and the following lysis treatment was performed.
(3) 10% (w / v) DBSA aqueous solution containing 12% (w / v) 2-propanol and 2.8 mg / mL MEGA-10 with respect to 200 μL of the bacterial solution prepared in (2) ( 200 μL each of neutralized with Tris) or sterilized distilled water was added and mixed.
(4) Of the liquids mixed in (3), the liquid mixed with the DBSA aqueous solution is heated at 70 ° C. or 80 ° C. for 7 minutes, 9 minutes, 10 minutes or 15 minutes, and the liquid mixed with distilled water for 10 minutes, respectively. did. After heating, it was left on ice for 5 minutes or more. The treatment was performed in duplicate at each temperature and time.
(5) After the heat treatment, the supernatant was removed by strong centrifugation, and then the 1/15 M phosphate buffer solution (hereinafter referred to as T-PB) containing 0.05% (w / v) Tween 80 (Sigma) in the precipitate. 1 mL) was added, and the precipitate was washed by stirring well.
(6) After washing (5) again, the precipitate was finally suspended in 300 μL of T-PB, and the entire amount was applied to 7H9-C plate medium (Kyokuto Pharmaceutical Co., Ltd.). The culture was performed under the conditions of 2 . The culture period was 8 weeks in accordance with the tuberculosis test guideline 2007 (edited by the Mycobacterium tuberculosis society mycobacteria test method review committee). In addition, for the purpose of confirming whether or not the bacterial solution diluted in (1) was correctly prepared to 10 7 cfu / mL, a solution obtained by serially diluting 10 5 times using T-PB from the bacterial solution was added to 7H9-C. What applied to the plate culture medium was culture | cultivated simultaneously as culture | cultivation positive.
(7) When colonies were observed, the number of colonies was counted. No colonies were found after 8 weeks. D. It was.
培養の結果を表７に示す。蒸留水を用いた場合、一部試料で菌の検出が確認され、バイオセーフティが必ずしも確保されていないことがわかる。一方、本発明の試薬を用いた場合は、いずれの試料も菌の検出が確認されないことから、少なくとも７０℃以上かつ７分間以上の加熱処理を行なえば、バイオセーフティが確保された溶菌操作が行なえるといえる。すなわち、前述した本発明の試薬による抗酸菌の処理方法は、濃厚試料や培養液が検体であったとしても、１０７ｃｆｕの抗酸菌を死滅させることができるため、バイオセーフティの観点からも極めて有効な方法といえる。 The culture results are shown in Table 7. When distilled water is used, the detection of bacteria is confirmed in some samples, and it can be seen that biosafety is not necessarily ensured. On the other hand, when the reagent of the present invention is used, detection of bacteria is not confirmed in any of the samples. Therefore, a lysis operation in which biosafety is ensured can be performed by performing a heat treatment of at least 70 ° C. and 7 minutes or more. It can be said. That is, the above-mentioned method for treating acid-fast bacteria with the reagent of the present invention can kill 10 7 cfu acid-fast bacteria even if a concentrated sample or a culture solution is a specimen. Is also an extremely effective method.
実施例１から７においては、強酸性のドデシルベンゼンスルホン酸（ＤＢＳＡ）水溶液の中和剤として、トリスヒドロキシメチルアミノメタン（Ｔｒｉｓ）を採用したが、アルキルベンゼンスルホン酸との反応により前記酸の金属塩を生成しない他の中和剤が採用できるか検討した。
（１）実施例１（１）から（２）に記載の方法にしたがい、１０２ｃｆｕのＢＣＧを含む１００μＬの菌液を調製した。
（２）（１）で調製した菌液１００μＬに、以下に示す（Ａ）から（Ｃ）の水溶液１００μＬを添加し混和後、８０℃で１０分加熱処理した。
（Ａ）中和剤としてＴｒｉｓを含む１０％（ｗ／ｖ）ＤＢＳＡ水溶液
（Ｂ）中和剤としてアンモニア水（ＮＨ３）を含む１０％（ｗ／ｖ）ＤＢＳＡ水溶液
（Ｃ）中和剤として２−アミノエタノール（ＮＨ２−ＥｔＯＨ）を含む１０％（ｗ／ｖ）ＤＢＳＡ水溶液
（３）実施例１（４）と同様な方法で精製し、実施例１（５）と同様な方法で陽性時間を測定した。
(1) According to the method described in Example 1 (1) to (2), 100 μL of a bacterial solution containing 10 2 cfu of BCG was prepared.
(2) To 100 μL of the bacterial solution prepared in (1), 100 μL of the aqueous solutions (A) to (C) shown below were added and mixed, followed by heat treatment at 80 ° C. for 10 minutes.
(A) 10% (w / v) DBSA aqueous solution containing Tris as neutralizing agent (B) 10% (w / v) DBSA aqueous solution containing ammonia water (NH 3 ) as neutralizing agent (C) As neutralizing agent 10% (w / v) DBSA aqueous solution containing 2 -aminoethanol (NH 2 -EtOH) (3) Purified by the same method as in Example 1 (4), positive by the same method as in Example 1 (5) Time was measured.
結果を表８に示す。本実施例で検討した、アルキルベンゼンスルホン酸との反応により前記酸の金属塩を生成しない中和剤は、いずれも使用可能であることがわかる。 The results are shown in Table 8. It can be seen that any of the neutralizing agents studied in this example that does not produce a metal salt of the acid by reaction with an alkylbenzene sulfonic acid can be used.
本発明の試薬を用いた抗酸菌を溶菌し核酸を抽出する方法は、特殊な装置を用いることなく、簡便かつ短時間に抗酸菌から核酸を確実に抽出でき、かつ操作者の安全（バイオセーフティ）が確保された方法である。したがって、当該方法を、例えば、核酸の増幅・検出法による抗酸菌検査における試料の前処理に適用することにより、安全かつ効率的な抗酸菌検査を容易に実現できる。 The method for lysing acid-fast bacteria using the reagent of the present invention and extracting nucleic acid can extract nucleic acid from acid-fast bacteria easily and in a short time without using a special device, and can be safe for the operator ( This is a method that ensures biosafety. Therefore, a safe and efficient acid-fast bacteria test can be easily realized by applying the method to, for example, pretreatment of a sample in an acid-fast bacilli test using a nucleic acid amplification / detection method.
Claims (8)
前記工程で抽出した抗酸菌由来ＲＮＡを当該ＲＮＡに特異的なプライマー・プローブを用いて、増幅・検出する工程とを含む、
試料中に含まれる抗酸菌を検出する方法。 After adding an aqueous solution containing an alkylbenzene sulfonic acid and a neutralizing agent that does not produce a metal salt of the acid by reaction with the acid to a sample containing acid-fast bacteria, the acid-fast bacteria are lysed by RNA treatment. Extracting the
Amplifying and detecting the mycobacteria-derived RNA extracted in the above step using a primer / probe specific to the RNA,
A method for detecting acid-fast bacteria contained in a sample.
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| JP2018510961A (en) * | 2015-04-30 | 2018-04-19 | ダウ グローバル テクノロジーズ エルエルシー | Amino alcohol salts of alkylbenzene sulfonic acids and their use in detergent formulations |
| WO2023286535A1 (en) * | 2021-07-15 | 2023-01-19 | 栄研化学株式会社 | Method for reducing nonspecific amplification in nucleic acid amplification reaction |
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| JP2017192350A (en) * | 2016-04-21 | 2017-10-26 | 東ソー株式会社 | Method for lysis of mycoplasma pneumoniae and method for detection thereof |
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