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Description
核酸を増幅する手法として、鎖置換活性を有するＤＮＡポリメラーゼを用いて遺伝子増幅反応を等温で進行させる方法（LAMP（Loop-mediated Isothermal Amplification）法）が開発されている（特許文献５）。この方法は、４種類のプライマーを用い、それらのうち２種類のインナープライマーは、それらの３’と５’側で標的核酸配列中の異なる２領域を認識し、５’側の配列はその３’側からの伸長反応で合成した相補鎖領域内にアニールするよう設計される。増幅反応は、これらのインナープライマーと、インナープライマーを起点に合成されたＤＮＡ鎖を鋳型ＤＮＡから一本鎖として剥がすために用いられる２種類のアウタープライマーにより生成する、ステムループ構造を持つダンベル型の構造を起点とし、自己伸長反応と鎖置換合成反応を繰り返すことで、進行する。この方法では、増幅反応は等温（60〜65°C）で行われる。
また、ダンベル構造の5'末端側のループの一本鎖部分に相補的な配列を持つ２種類のループプライマーを用いることにより、ＤＮＡ合成の起点を増やし、反応時間を短縮することが可能となる（特許文献６）。
As a method for amplifying a nucleic acid, a method (LAMP (Loop-mediated Isothermal Amplification) method) in which a gene amplification reaction proceeds isothermally using a DNA polymerase having strand displacement activity has been developed (Patent Document 5). This method uses four types of primers, and two of them recognize two different regions in the target nucleic acid sequence on the 3 ′ and 5 ′ sides, and the sequence on the 5 ′ side is the 3 It is designed to anneal in the complementary strand region synthesized by the extension reaction from the side. The amplification reaction dumbbell with the these inner primer, generated by two outer primer that is used to peel the DNA strand synthesized inner primer as a starting point, as a single strand from the template DNA, a stem-loop structure It proceeds by repeating the self-elongation reaction and the strand displacement synthesis reaction starting from the structure of In this method, the amplification reaction is performed isothermally (60-65 ° C).
In addition, by using two types of loop primers having sequences complementary to the single-stranded portion of the loop on the 5 ′ end side of the dumbbell structure, it becomes possible to increase the starting point of DNA synthesis and shorten the reaction time. (Patent Document 6).
すなわち本発明は、被検試料中の微生物の生細胞を、死細胞及び／又は損傷細胞と識別して検出する方法であって、以下の工程：
ａ）前記被検試料に、微生物の核酸の核酸増幅法による増幅を、死細胞に選択的に阻害する薬剤を添加する工程、
ｂ）微生物の細胞の透過性を高める処理を行う工程、
ｃ）被検試料中の微生物の核酸のターゲット領域を、細胞からの核酸の抽出を行わずに、鎖置換型核酸伸長酵素を用いた等温核酸増幅法により増幅する工程、及び
ｄ）増幅産物を解析する工程、
を含み、前記微生物の細胞の透過性を高める処理は、それによって細胞からの核酸の抽出を行わずに、生細胞に選択的な核酸増幅を可能にする処理である、方法を提供する。
That is, the present invention is a method for detecting living cells of microorganisms in a test sample by distinguishing them from dead cells and / or damaged cells, and the following steps:
a) a step of adding, to the test sample, an agent that selectively inhibits the dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
b) performing a treatment for increasing the permeability of cells of microorganisms;
c) a step of amplifying a target region of a nucleic acid of a microorganism in a test sample by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme without extracting the nucleic acid from the cell, and d) an amplification product Process to analyze,
And the process of increasing the permeability of the microorganism cell is thereby a process that allows selective nucleic acid amplification in living cells without the extraction of nucleic acids from the cells.
また、本発明は、鎖置換型核酸伸長酵素を用いた等温核酸増幅法により、被検試料中の微生物の生細胞を、死細胞及び／又は損傷細胞と識別して検出するためのキットであって、下記の要素を含むキットを提供する：
１）微生物の核酸の核酸増幅法による増幅を、死細胞に選択的に阻害する薬剤、
２）下記組成の反応液を調製するための試薬、
Tris-HCl(pH7〜9) 10mM〜25mM
KCl 5mM〜15mM
MgSO4 5mM〜40mM
界面活性剤 0.1%〜0.4%
ベタイン 0.5M〜1M
dNTPs 各1mM〜1.5mM
３）検出対象の微生物の核酸のターゲット領域を等温核酸増幅法により増幅するためのプライマー。
Further, the present invention is a kit for distinguishing and detecting living cells of microorganisms in a test sample from dead cells and / or damaged cells by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme. Provides a kit containing the following elements:
1) a drug that selectively inhibits dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
2) a reagent for preparing a reaction solution having the following composition;
Tris-HCl (pH 7 ~ 9) 10mM ~ 25mM
KCl 5mM ~ 15mM
MgSO 4 5mM~40mM
Surfactant 0.1% -0.4%
Betaine 0.5M-1M
dNTPs 1mM to 1.5mM each
3) A primer for amplifying the target region of the nucleic acid of the microorganism to be detected by an isothermal nucleic acid amplification method.
また本発明は、被検試料中の微生物の生細胞を、死細胞及び／又は損傷細胞と識別して検出する方法であって、以下の工程：
ａ）前記被検試料に、微生物の核酸の核酸増幅法による増幅を、死細胞に選択的に阻害する薬剤を添加する工程、
ｃ）被検試料中の微生物の核酸のターゲット領域を核酸増幅法により増幅する工程、及びｄ）増幅産物を解析する工程、
を含む方法において、工程ａ）を蛋白質、糖類、脂質、及び酵母エキスからなる群から選択される成分を０．５〜１０質量％含む細胞懸濁液中で行うことを特徴とする方法を提供する。
The present invention also relates to a method for detecting a living cell of a microorganism in a test sample by distinguishing it from a dead cell and / or a damaged cell, the following steps:
a) a step of adding, to the test sample, an agent that selectively inhibits the dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
c) a step of amplifying a target region of a nucleic acid of a microorganism in a test sample by a nucleic acid amplification method, and d) a step of analyzing an amplification product,
A process comprising performing a) in a cell suspension containing 0.5 to 10% by mass of a component selected from the group consisting of proteins, sugars, lipids, and yeast extracts To do.
本発明の方法によれば、簡便に、微生物の生細胞を、死細胞及び／又は損傷細胞と識別して検出することができる。本発明により、核酸増幅法による簡易かつ迅速な食品及び生体試料、拭き取り試料、工業用水、環境用水、排水等の環境中の微生物の生細胞・損傷細胞・死細胞の判別が可能となる。本発明の方法及びキットは、自主検査に応用可能であり、経済性にも優れている。 According to the method of the present invention, living cells of microorganisms can be detected by distinguishing them from dead cells and / or damaged cells. According to the present invention, it is possible to discriminate between living cells, damaged cells, and dead cells of microorganisms in an environment such as food and biological samples, wiped samples, industrial water, environmental water, wastewater, and the like by a nucleic acid amplification method. The method and kit of the present invention can be applied to self-inspection and are excellent in economy.
＜１＞本発明の方法
本発明の方法は、被検試料中の微生物の生細胞を、死細胞及び／又は損傷細胞と識別して検出する方法であって、以下の工程：
ａ）前記被検試料に、微生物の核酸の核酸増幅法による増幅を、死細胞に選択的に阻害する薬剤を添加する工程、
ｂ）微生物の細胞の透過性を高める処理を行う工程、
ｃ）被検試料中の微生物の核酸のターゲット領域を、細胞からの核酸の抽出を行わずに、鎖置換型核酸伸長酵素を用いた等温核酸増幅法により増幅する工程、及び
ｄ）増幅産物を解析する工程、
を含む。前記微生物の細胞の透過性を高める処理は、それによって細胞からの核酸の抽出を行わずに、生細胞に選択的な核酸増幅を可能にする処理である。
<1> Method of the Present Invention The method of the present invention is a method for detecting living cells of microorganisms in a test sample by distinguishing them from dead cells and / or damaged cells, and the following steps:
a) a step of adding, to the test sample, an agent that selectively inhibits the dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
b) performing a treatment for increasing the permeability of cells of microorganisms;
c) a step of amplifying a target region of a nucleic acid of a microorganism in a test sample by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme without extracting the nucleic acid from the cell, and d) an amplification product Process to analyze,
including. The treatment for increasing the permeability of the cells of the microorganism is a treatment that enables nucleic acid amplification selective to living cells without extracting nucleic acids from the cells.
また、生体試料としては、血液試料、尿試料、髄液試料、滑液試料、胸水試料、喀痰試料、糞便試料、鼻腔粘液試料、喉頭粘液試料、胃洗浄液試料、膿汁試料、皮膚粘膜試料、口腔粘液試料、呼吸器粘膜試料、消化器粘膜試料、眼結膜試料、胎盤試料、生殖細胞試料、産道試料、母乳試料、唾液試料、嘔吐物、又は水疱内容物等が例示される。
さらに、環境用水としては、市水、地下水、河川水、又は雨水等が例示される。
Biological samples include blood samples, urine samples, spinal fluid samples, synovial fluid samples, pleural effusion samples, sputum samples, stool samples, nasal mucus samples, laryngeal mucus samples, gastric lavage fluid samples, pus juice samples, skin mucosa samples, oral cavity Examples include mucus samples, respiratory mucosa samples, digestive mucosa samples, eye conjunctiva samples, placenta samples, germ cell samples, birth canal samples, breast milk samples, saliva samples, vomiting, blister contents , and the like.
Furthermore, examples of the environmental water include city water, ground water, river water, and rain water.
特に、食品衛生検査や臨床検査において、穏和な加熱処理や抗生物質投与により、損傷細胞の状態を呈した細菌の検出が注目されており、本発明は、生細胞の検出のみならず、生細胞と死細胞及び/又は損傷細胞との識別も可能な微生物の検出方法を提供するものである。 In particular, in food sanitation inspection and clinical test, the mild heat treatment or administration of antibiotics, attention has been focused on the detection of bacteria exhibiting the state of injured cell, the onset Ming, not only detection of live cells, live It is an object of the present invention to provide a method for detecting microorganisms that can distinguish cells from dead cells and / or damaged cells.
本発明において、「生細胞の検出」とは、被検試料中の生細胞の有無の判別及び生細胞の量の決定のいずれをも含む。また、生細胞の量とは、絶対的な量に限られず、対照試料に対する相対的な量であってもよい。また、「生細胞を、死細胞及び／又は損傷細胞と識別して検出する」とは、死細胞及び／又は損傷細胞に比べて選択的に検出することを意味する。 In the present invention, “detection of living cells” includes both determination of presence / absence of living cells in the test sample and determination of the amount of living cells. Further, the amount of living cells is not limited to an absolute amount, and may be an amount relative to a control sample. Further, “detecting a living cell by distinguishing it from a dead cell and / or a damaged cell” means that the cell is selectively detected as compared with a dead cell and / or a damaged cell .
先の薬剤処理と、それ以降の薬剤処理との間で、未反応の薬剤を除去する工程を追加してもよい。薬剤を除去する方法としては、被検試料を遠心分離して、微生物を含む沈殿と薬剤を含む上清とを分離し、上清を除去する方法が挙げられる。この場合、薬剤を除去した後、適宜、洗浄剤で微生物を洗浄する工程を追加することも可能である。 A step of removing unreacted drug may be added between the previous drug process and the subsequent drug process . Examples of the method for removing the drug include a method of centrifuging a test sample, separating a precipitate containing a microorganism and a supernatant containing a drug, and removing the supernatant. In this case, it is possible to add a step of washing the microorganism with a cleaning agent as appropriate after removing the drug.
以上のことから、工程ｂ）は、好ましくは細胞の形態を保ちつつ、等温核酸増幅反応に必要な試薬を微生物の細胞内に透過させるための工程と言い換えることができる。すなわち、「細胞の透過性」（transparency of cell）とは、等温核酸増幅反応に必要な試薬が生物の細胞内に透過する性質（permeability）を意味する。微生物の細胞の透過性を高める処理は、微生物の細胞壁及び／又は細胞膜を穿孔する処理、又は、細胞壁及び／又は細胞膜の構造を緩める処理であってもよい。 From the above, step b) can be rephrased as a step for permeating the reagent necessary for the isothermal nucleic acid amplification reaction into the cells of the microorganism, preferably while maintaining the cell morphology. That is, the "cell permeability" (transparency of cel l), reagents necessary for isothermal nucleic acid amplification reaction refers to properties (permeability) which passes through the cells of the organism. The treatment for increasing the permeability of the cells of the microorganism may be a treatment for perforating the cell wall and / or cell membrane of the microorganism or a treatment for loosening the structure of the cell wall and / or cell membrane.
（３）工程ｃ）
続いて、被検試料中の微生物の核酸のターゲット領域を、細胞からの核酸の抽出を行わずに、鎖置換型核酸伸長酵素を用いた等温核酸増幅法により増幅する。
(3) Step c)
Subsequently, the target region of the nucleic acid of the microorganism in the test sample is amplified by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme without extracting the nucleic acid from the cell.
また、等温核酸増幅法のうちＲＮＡをターゲットとする方法としては、逆転写酵素によりコンプリメンタリＤＮＡ（cDNA）を合成し、さらに逆転写酵素のＤＮＡポリメラーゼ活性により二本鎖ＤＮＡを合成し、最終的にＲＮＡポリメラーゼにより、ＲＮＡの特異的領域のみを増幅するＴＲＣ法（Nakaguchi, Y. et al., J. Clin. Microbiol., 42(9):4284-4292, 2004）が例示される。ＴＲＣ法では、増幅産物はＲＮＡである。 In the isothermal nucleic acid amplification method, RNA is targeted by synthesizing complementary DNA (cDNA) by reverse transcriptase, and further by synthesizing double-stranded DNA by the DNA polymerase activity of reverse transcriptase. The TRC method (Nakaguchi, Y. et al., J. Clin. Microbiol., 42 (9): 4284-4292, 2004) in which only a specific region of RNA is amplified by RNA polymerase is exemplified. In the TRC method, the amplification product is RNA.
＜２＞本発明のキット
本は発明のキットは、鎖置換型核酸伸長酵素を用いた等温核酸増幅法により、被検試料中の微生物の生細胞を、死細胞及び／又は損傷細胞と識別して検出するためのキットであって、下記の要素を含む。
１）微生物の核酸の核酸増幅法による増幅を、死細胞に選択的に阻害する薬剤、
２）下記組成の反応液を調製するための試薬、
Tris-HCl(pH7〜9) 10mM〜25mM
KCl 5mM〜15mM
MgSO4 5mM〜40mM
界面活性剤 0.1%〜0.4%
ベタイン 0.5M〜1M
dNTPs 各1mM〜1.5mM
鎖置換型核酸伸長酵素 0.2〜0.6U/μl
３）検出対象の微生物の核酸のターゲット領域を等温核酸増幅法により増幅するためのプライマー。
<2> Kit of the Present Invention The kit of the present invention discriminates living cells of microorganisms in test samples from dead cells and / or damaged cells by isothermal nucleic acid amplification using a strand displacement type nucleic acid elongation enzyme. And a kit for detecting the following.
1) a drug that selectively inhibits dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
2) a reagent for preparing a reaction solution having the following composition;
Tris-HCl (pH 7 ~ 9) 10mM ~ 25mM
KCl 5mM ~ 15mM
MgSO 4 5mM~40mM
Surfactant 0.1% -0.4%
Betaine 0.5M-1M
dNTPs 1mM to 1.5mM each
Strand displacement type nucleic acid elongation enzyme 0.2-0.6 U / μl
3) A primer for amplifying the target region of the nucleic acid of the microorganism to be detected by an isothermal nucleic acid amplification method.
すなわち、本発明は、被検試料中の微生物の生細胞を、死細胞及び／又は損傷細胞と識別して検出する方法であって、以下の工程：
ａ）前記被検試料に、微生物の核酸の核酸増幅法による増幅を、死細胞に選択的に阻害する薬剤を添加する工程、
ｃ）被検試料中の微生物の核酸のターゲット領域を核酸増幅法により増幅する工程、及びｄ）増幅産物を解析する工程、
を含む方法において、工程ａ）を酵母エキスを０．５％〜１０％含む細胞懸濁液中で行うことを特徴とする方法を提供する。
That is, the present invention is a method for detecting living cells of microorganisms in a test sample by distinguishing them from dead cells and / or damaged cells, and the following steps:
a) a step of adding, to the test sample, an agent that selectively inhibits the dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
c) a step of amplifying a target region of a nucleic acid of a microorganism in a test sample by a nucleic acid amplification method, and d) a step of analyzing an amplification product,
A method is provided wherein step a) is performed in a cell suspension containing 0.5 % to 10 % yeast extract.
まず、対照群（コントロール）となる基本マスターミックス組成を表５に示す。この基本マスターミックスは、栄研化学 レジオネラ検出試薬キットEの基本組成に、このキットの添付文書に従いカルセインを添加したものであるが、反応容量を1/2にスケールダウンさせ、11 μlのマスターミックスに2.5 μl相当のDNA溶液を鋳型として添加した反応系である。 First, the basic master mix composition used as a control group (control) is shown in Table 5. This basic master mix is the basic composition of Eiken Chemical Legionella Detection Reagent Kit E with calcein added according to the package insert of this kit. 2.5 [mu] l equivalent of DNA solution is a reaction system was added as a template to.
前記全ての遺伝子情報を一列に並べ、ClustalWにより各遺伝子領域を解析し、前記全ての属で一致した塩基、及び、一つでも完全一致性が保たれなかった塩基を同定し、各微生
物の16S rRNA遺伝子領域中の保存領域とバリアント領域を解析した。
Arranging said all genetic information in a row, analyzes each gene region by ClustalW, the all matching bases in the genus, and to identify one Tsude also completely matching is not maintained bases, for each microorganism The conserved region and variant region in the 16S rRNA gene region were analyzed.
表２４によれば、対照群に相当する生理食塩水にけん濁させたレジオネラ生細胞は、細胞濃度2.4 log cfu/ml（107希釈）、及び、3.4 log cfu/ml（106希釈）では、陰性と判定される結果となったが、7.4 log cfu/ml（102倍希釈）では、EMA処理後でも陽性と判定された。したがって、細胞濃度が高ければ、生理食塩水懸濁液でEMA処理した場合でも、生細胞・死細胞の識別が可能であることが示された。 According to Table 24, the live Legionella cells suspended in the physiological saline corresponding to the control group had the cell concentrations of 2.4 log cfu / ml (10 7 dilution) and 3.4 log cfu / ml (10 6 dilution). The result was determined to be negative, but it was determined to be positive even after EMA treatment at 7.4 log cfu / ml (10 2 fold dilution). Therefore, it was shown that if the cell concentration is high, it is possible to distinguish between live and dead cells even when EMA treatment is performed with a physiological saline suspension.
尚、Y-SWとYS-SWの死細胞の各結果を比較することにより、SR110（ACES/水酸化カリウムバッファー、ピロリン酸第二鉄、L-システイン塩酸塩、α-ケトグルタル酸）という本来レジオネラ生細胞の発育にとって必須の成分が、EMAのレジオネラ死細胞への透過を阻害するか、又はそれらの成分がプラスチャージを帯びているEMAと結合しEMAを不活性化させた可能性が示唆される。 By comparing the results of each of the dead cells of Y-SW and YS-SW, SR110 ( ACES / potassium hydroxide buffer, ferric pyrophosphate, L-cysteine hydrochloride, α-ketoglutarate ) and It is possible that components essential for the growth of Legionella live cells inhibit EMA permeation into Legionella dead cells, or these components bind to EMA with a positive charge and inactivate EMA. Is suggested.
７−１）試験方法
Legionella pneumophila ATCC33153株をBCYEα培地にて37℃、2日培養したコロニーを釣菌し、滅菌した1% Bacto Yeast Extract水溶液（Y-SW）にけん濁させ、8.9 log cfu/mlの生細胞けん濁液を調製した。その後、Y-SWにて102倍希釈けん濁液を調製し、10分煮沸して死細胞けん濁液を調製した。また、前記8.9 log cfu/mlの生細胞けん濁液の102〜108倍希釈けん濁液をY-SWにより調製した。
上記の各生細胞懸濁液及び死細胞けん濁液1 mlを試験検体とし、EMA 25〜35 μg/mlの終濃度にて計3回の多段階EMA処理（１回目EMA処理 遮光下氷上35 μg/ml 15分、２回目EMA処理30 μg/ml 15分、３回目EMA処理 25 μg/ml 10分）を行った。各EMA処理後の可視光照射は10分とした。また、それぞれの検体に対し未処理群も用意した。
7-1) Test method
Legionella pneumophila ATCC33153 strain was cultured in BCYEα medium at 37 ° C for 2 days, colonies were picked, suspended in sterile 1% Bacto Yeast Extract aqueous solution (Y-SW), and 8.9 log cfu / ml live cell suspension A liquid was prepared. Thereafter, a suspension diluted 10 2 times with Y-SW was prepared and boiled for 10 minutes to prepare a dead cell suspension. In addition, a 10 2 to 10 8 times diluted suspension of the 8.9 log cfu / ml live cell suspension was prepared by Y-SW.
Using each of the above live cell suspensions and 1 ml of dead cell suspension as a test sample, a total of 3 multi-stage EMA treatments (1st EMA treatment on light-shielded ice 35) at a final concentration of EMA 25-35 μg / ml μg / ml 15 minutes, second EMA treatment 30 μg / ml 15 minutes, third EMA treatment 25 μg / ml 10 minutes). Visible light irradiation after each EMA treatment was 10 minutes. An untreated group was also prepared for each specimen.
表２５及び表２６における記号は以下のとおりである。
a) L. pneumophila ATCC33153のY-SW、又は生理食塩水けん濁液(8.9 ±0.31 log cells/ml)
b) L. pneumophila ATCC33153の死細胞懸濁液（6.9 ±0.31 log cells/ml）、又は、8.9 log cells/mlの生細胞けん濁液をY-SW、もしくは、生理食塩水により102倍希釈し生細胞
けん濁液。
c) LAMP増幅は２回実施し、Ct値（サイクル数、又はmin）をMean±SD (n = 2)として記載する。
d) ２回の結果がLAMP未増幅（陰性）を示す。
The symbols in Table 25 and Table 26 are as follows.
a) Y.SW of L. pneumophila ATCC33153 or saline suspension (8.9 ± 0.31 log cells / ml)
b) dead cell suspension L. pneumophila ATCC33153 (6.9 ± 0.31 log cells / ml), or, 8.9 log cells / ml of live cell-suspension liquid Y-SW or, 10 2-fold diluted with physiological saline Live cell suspension.
c) Perform LAMP amplification twice and describe the Ct value (number of cycles or min) as Mean ± SD (n = 2).
d) Two results indicate LAMP unamplified (negative).
〔実施例８〕多段階EMA処理−ダイレクトLAMP法によるレジオネラ生細胞の一斉検出
本実施例では、ダイレクトLAMP法による様々なレジオネラ菌の生細胞の検出限界を検討した。
Example 8 multistage EMA treatment - The simultaneous detection embodiment of Legionella cells by direct LAMP method, the detection limit of live cells of a variety of Legionella by direct LAMP method was consider.
Claims (3)
ａ）前記被検試料に、微生物の核酸の核酸増幅法による増幅を、死細胞に選択的に阻害する薬剤を添加する工程、
ｂ）微生物の細胞の透過性を高める処理を行う工程、
ｃ）被検試料中の微生物の核酸のターゲット領域を、細胞からの核酸の抽出を行わずに、鎖置換型核酸伸長酵素を用いた等温核酸増幅法により増幅する工程、及び
ｄ）増幅産物を解析する工程、
を含み、前記微生物の細胞の透過性を高める処理は、それによって細胞からの核酸の抽出を行わずに、生細胞に選択的な核酸増幅を可能にする処理である、方法。 A method for detecting living cells of microorganisms in a test sample by distinguishing them from dead cells and / or damaged cells, comprising the following steps:
a) a step of adding, to the test sample, an agent that selectively inhibits the dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
b) performing a treatment for increasing the permeability of cells of microorganisms;
c) a step of amplifying a target region of a nucleic acid of a microorganism in a test sample by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme without extracting the nucleic acid from the cell, and d) an amplification product Process to analyze,
Wherein the treatment of increasing the permeability of the cells of the microorganism is a treatment thereby enabling nucleic acid amplification selective to living cells without extraction of nucleic acids from the cells.
１）微生物の核酸の核酸増幅法による増幅を、死細胞に選択的に阻害する薬剤、
２）下記組成の反応液を調製するための試薬、
Tris-HCl(pH7〜9) 10mM〜25mM
KCl 5mM〜15mM
MgSO4 5mM〜40mM
界面活性剤 0.1%〜0.4%
ベタイン 0.5M〜1M
dNTPs 各1mM〜1.5mM
３）検出対象の微生物の核酸のターゲット領域を等温核酸増幅法により増幅するためのプライマー。 A kit for detecting a living cell of a microorganism in a test sample by distinguishing it from a dead cell and / or a damaged cell by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme, comprising: Including kit:
1) a drug that selectively inhibits dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
2) a reagent for preparing a reaction solution having the following composition;
Tris-HCl (pH 7 ~ 9) 10mM ~ 25mM
KCl 5mM ~ 15mM
MgSO 4 5mM~40mM
Surfactant 0.1% -0.4%
Betaine 0.5M-1M
dNTPs 1mM to 1.5mM each
3) A primer for amplifying the target region of the nucleic acid of the microorganism to be detected by an isothermal nucleic acid amplification method.
ａ）前記被検試料に、微生物の核酸の核酸増幅法による増幅を、死細胞に選択的に阻害する薬剤を添加する工程、
ｃ）被検試料中の微生物の核酸のターゲット領域を核酸増幅法により増幅する工程、及びｄ）増幅産物を解析する工程、
を含む方法において、工程ａ）を蛋白質、糖類、脂質、及び酵母エキスからなる群から選択される成分を０．５〜１０％含む細胞懸濁液中で行うことを特徴とする方法。 A method for detecting living cells of microorganisms in a test sample by distinguishing them from dead cells and / or damaged cells, comprising the following steps:
a) a step of adding, to the test sample, an agent that selectively inhibits the dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
c) a step of amplifying a target region of a nucleic acid of a microorganism in a test sample by a nucleic acid amplification method, and d) a step of analyzing an amplification product,
A step comprising performing step a) in a cell suspension containing 0.5 to 10% of a component selected from the group consisting of proteins, sugars, lipids, and yeast extracts.
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