JP2010046016A - Method for microorganism detection - Google Patents
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Abstract
Description
本発明は、食品などの被検試料中に存在する微生物の生菌を検出・同定する方法に関する。 The present invention relates to a method for detecting and identifying viable microorganisms present in a test sample such as food.
食品中に存在する食中毒菌や腐敗菌等の微生物や、環境中に存在する各種微生物の生菌の有無を確認することは、食品の品質管理や感染症の防止などのために重要である。上記のような被検試料中に存在する微生物を確認・同定する一般的な方法として、従来よりシャーレ等に作製した固体培地上に被検試料を置き、数日間培養して菌を培地上に生育させ、その形体等から微生物の種類を確認、同定する平板培養などの培養法が用いられてきたが、これに加えて、遺伝子解析技術の進歩を背景として特に近年は微生物の検出及び同定をより短時間で行うための方法の開発も進んでいる。
微生物を短時間で検出する一般的な方法としてＰＣＲ法により微生物のＤＮＡを増幅して検出する方法があるが、被検試料に対して単純にこの方法を用いただけでは生菌のみならず死菌のＤＮＡも増幅されてしまうため、偽陽性となる可能性がある。この問題の解決のために、生菌と死菌を区別して検出する技術も報告されており、例えば特開２００８−６７７１８号公報には、死菌および損傷菌のＤＮＡを選択的に切断する条件下で死菌及び損傷菌のＤＮＡを酵素で切断し、リアルタイムＰＣＲによって生菌の遺伝子を増幅して解析する方法が開示されている（特許文献１）。さらに、被検試料を増菌用液体培地で培養し、０時間目と４時間目の被検試料の核酸の量の違いをリアルタイムＰＣＲで検出することによって生菌の存在を確認する方法も報告されている（http://www.amr-inc.jp/detail/ez-cocktail.html：非特許文献１）。
しかしながら、上記のようにリアルタイムＰＣＲを用いる方法では、ＰＣＲ時に使用できるプライマー数や検出できるシグナルの数などの制限により、数多くの微生物種を高感度に検出するのには限界があり、また、増幅された微生物の種類の同定は同時に行うことはできず、シークエンス等の遺伝子解析を行って微生物を１種ずつ同定する作業がさらに必要となるなどの問題点があった。従って、食品などの被検試料中に存在する微生物の生菌を迅速かつ簡便に検出し、かつ同時に微生物の種類を同定することが可能な技術の開発が必要とされていた。
It is important for food quality control, prevention of infectious diseases, etc. to confirm the presence or absence of microorganisms such as food poisoning bacteria and spoilage bacteria present in food and the presence of live microorganisms of various microorganisms present in the environment. As a general method for confirming and identifying the microorganisms present in the test sample as described above, the test sample is placed on a solid medium conventionally prepared in a petri dish or the like, and cultured for several days to put the bacteria on the medium. Culture methods such as plate culture have been used for growing and confirming and identifying the type of microorganism from its form, etc.In addition to this, in recent years the detection and identification of microorganisms have been especially advanced against the background of genetic analysis technology. Development of a method for performing in a shorter time is also progressing.
As a general method for detecting microorganisms in a short period of time, there is a method of amplifying and detecting the DNA of microorganisms by the PCR method, but if this method is simply used for a test sample, not only live bacteria but also dead bacteria Is also amplified, which may result in false positives. In order to solve this problem, a technique for distinguishing and detecting live bacteria and dead bacteria has also been reported. For example, Japanese Patent Application Laid-Open No. 2008-67718 discloses conditions for selectively cleaving dead and damaged bacteria DNA. A method is disclosed in which DNA of dead and damaged bacteria is cleaved with an enzyme, and a gene of live bacteria is amplified and analyzed by real-time PCR (Patent Document 1). In addition, a method for confirming the presence of viable bacteria by culturing a test sample in a liquid medium for enrichment and detecting the difference in the amount of nucleic acid in the test sample at 0 and 4 hours by real-time PCR is also reported. (Http://www.amr-inc.jp/detail/ez-cocktail.html: Non-Patent Document 1).
However, in the method using real-time PCR as described above, there are limits to detecting many microorganism species with high sensitivity due to limitations such as the number of primers that can be used during PCR and the number of signals that can be detected. Identification of the types of microorganisms thus performed cannot be performed at the same time, and there is a problem that it is necessary to further identify the microorganisms one by one by performing gene analysis such as sequencing. Therefore, it has been necessary to develop a technique capable of quickly and easily detecting a living microbe in a test sample such as food and simultaneously identifying the type of the microbe.
本願発明者らは、食品等の試料をインキュベーションし、インキュベーション中の異なる２点の時間における核酸の量を、検出する微生物に特異的な検出対象領域とハイブリダイズし得るプローブが固定化された担体を用いて測定することにより、検出対象の微生物の生菌を迅速かつ簡便に検出し、同時に微生物の種類を同定できることを見出し、本願発明に至った。特に、インキュベーション中の異なる２点の時間でサンプリングした試料から核酸を抽出し、前記核酸を増幅し、検出対象である各微生物に特異的な検出対象領域を検出することにより、生死菌の判定および微生物種の同定が可能となる。 The inventors of the present application incubate a sample such as food, and a carrier on which a probe capable of hybridizing with a detection target region specific to a microorganism to detect the amount of nucleic acid at two different times during incubation is immobilized. As a result, it was found that viable bacteria of the microorganism to be detected can be detected quickly and easily, and at the same time, the type of the microorganism can be identified. In particular, a nucleic acid is extracted from samples sampled at two different times during incubation, the nucleic acid is amplified, and a detection target region specific to each microorganism to be detected is detected. Identification of microbial species becomes possible.
即ち、本発明は、
(a) 被検試料を、前記試料中に含まれる可能性のある微生物が増殖し得る条件下でインキュベーションする工程、
(b) 前記インキュベーションの時間ｔ0およびｔ1（ｔ1＞ｔ0）における前記試料から核酸を抽出する工程、
(c) 時間ｔ0およびｔ1において抽出した前記それぞれの核酸を鋳型として、検出対象とする微生物に特異的な検出対象領域を含む核酸配列を増幅させ、それぞれ増幅核酸試料ａ0および増幅核酸試料ａ1を得る工程、
(d) 増幅核酸試料ａ0および増幅核酸試料ａ1を、前記検出対象領域にハイブリダイズ可能なプローブが固定化された担体に接触させる工程、および
(e) 増幅核酸試料ａ0について前記担体に固定化されたプローブにハイブリダイズした核酸の量と、増幅核酸試料ａ1について前記担体に固定化されたプローブにハイブリダイズした核酸の量を比較する工程、
を含む、被検試料中の微生物の生菌を検出する方法を提供する。
That is, the present invention
(a) incubating the test sample under conditions that allow microorganisms that may be contained in the sample to grow;
(b) extracting nucleic acid from the sample at the incubation times t 0 and t 1 (t 1 > t 0 );
(c) Using each of the nucleic acids extracted at times t 0 and t 1 as a template, a nucleic acid sequence including a detection target region specific to the detection target microorganism is amplified, and an amplified nucleic acid sample a 0 and an amplified nucleic acid sample, respectively obtaining a 1 ,
(d) contacting the amplified nucleic acid sample a 0 and the amplified nucleic acid sample a 1 with a carrier on which a probe capable of hybridizing to the detection target region is immobilized; and
(e) Compare the amount of nucleic acid hybridized to the probe immobilized on the carrier for the amplified nucleic acid sample a 0 and the amount of nucleic acid hybridized to the probe immobilized to the carrier for the amplified nucleic acid sample a 1 Process,
A method for detecting viable microorganisms in a test sample is provided.
本発明の方法によれば、被検試料中の微生物の生菌を簡便かつ迅速に検出し、かつ同時に微生物の種類を同定することができる。 According to the method of the present invention, viable microorganisms in a test sample can be detected easily and quickly, and at the same time, the type of microorganism can be identified.
以下、本発明を詳細に説明する。
本発明の方法の工程(a)では、被検試料を、前記試料中に含まれる可能性のある微生物が増殖し得る条件下でインキュベーションする。本明細書において、被検試料は特に限定されず、検出対象とする微生物の生菌の存在の有無を確認する必要のあるものであればいずれのものでもよい。例えば、牛乳、水、緑茶、酸性飲料等の飲料、食肉、チルド食品、生鮮食品（肉、野菜、魚）、弁当・惣菜などの食品、および、食品製造環境中の空気などの環境試料が挙げられる。また、本明細書において「微生物」とは、本発明の方法を実施する者が検出することを所望するものであればいずれのものでもよく、例えば大腸菌、赤痢菌、サルモネラ菌、セレウス菌、キャンピロバクター、黄色ブドウ球菌、リステリア菌、腸炎ビブリオ菌、コレラ菌、ウェルシュ菌などの食中毒菌や、例えば、Aeromonas属、Bacillus属、Lactobacillus属、Pseudomonas属、Streptococcus属等の腐敗菌が挙げられる。
Hereinafter, the present invention will be described in detail.
In step (a) of the method of the present invention, the test sample is incubated under conditions that allow growth of microorganisms that may be contained in the sample. In the present specification, the test sample is not particularly limited, and any sample may be used as long as it is necessary to confirm the presence or absence of the living microorganism of the microorganism to be detected. Examples include beverages such as milk, water, green tea, acidic beverages, meat, chilled foods, fresh foods (meat, vegetables, fish), foods such as bento and side dishes, and environmental samples such as air in the food production environment. It is done. In the present specification, the term “microorganism” may be any microorganism as long as it is desired to be detected by a person who performs the method of the present invention. For example, Escherichia coli, Shigella, Salmonella, Cereus, Campylo Examples include food poisoning bacteria such as Bacter, Staphylococcus aureus, Listeria monocytogenes, Vibrio parahaemolyticus, Vibrio cholerae, and Clostridium perfringens, and spoilage bacteria such as Aeromonas , Bacillus , Lactobacillus , Pseudomonas and Streptococcus .
被検試料のインキュベーションの様式は、被検試料や検出対象とする微生物の種類に応じて当業者が適宜設定することが可能である。例えば被検試料が飲料等の液体である場合は、前記飲料を例えば孔径0.22 mmまたは0.45 mmのメンブレンフィルターにより吸引ろ過したものを液体培地でインキュベーションしてもよく、また被検試料が食品等の固形物である場合には、前記固形物を生理食塩水または適切な増菌培地に入れ、機械的に破砕したものをそのままインキュベーションしてもよい。検出対象とする微生物が増殖し得るインキュベーション条件も、同様に微生物の種類等に応じて当業者が適宜設定することが可能であり、例えば、細菌用として標準培地、SCD培地、BHI培地、真菌用としてポテトデキストロース培地などの汎用の液体培地を使用して３０〜６０℃の温度条件下、必要により振とうしながらインキュベーションすることができる。なお、本発明においては、操作の簡便性を考慮してインキュベーションを単一の培地中で行うこともでき、そのような培地の組成は検出対象とする微生物の種類を考慮して当業者が決定することが可能である。 The mode of incubation of the test sample can be appropriately set by those skilled in the art depending on the test sample and the type of microorganism to be detected. For example, when the test sample is a liquid such as a beverage, the beverage obtained by suction filtration using a membrane filter having a pore size of 0.22 mm or 0.45 mm, for example, may be incubated in a liquid medium. In the case of a solid material, the solid material may be placed in physiological saline or an appropriate enrichment medium, and the mechanically crushed material may be incubated as it is. Similarly, incubation conditions under which microorganisms to be detected can grow can be appropriately set by those skilled in the art according to the type of microorganism, for example, for bacteria, standard medium, SCD medium, BHI medium, fungus Incubation can be performed using a general-purpose liquid medium such as potato dextrose medium at 30 to 60 ° C. with shaking if necessary. In the present invention, the incubation can be performed in a single medium in consideration of the ease of operation, and the composition of such a medium is determined by those skilled in the art in consideration of the type of microorganism to be detected. Is possible.
さらに、上記のインキュベーションの条件は、検出対象とする微生物の死菌が被検試料中に存在する場合に前記死菌の核酸量が培養時間の経過とともに減少するものであることが好ましい。このような条件下でインキュベーションすることにより、インキュベーション時間の経過に伴う生菌と死菌の核酸量の変化を区別し、この核酸量の変化の仕方に基づいて後の工程により生菌と死菌を判定することが可能となる。このような条件は当技術分野において数多く知られており、例えばＲＮＡの場合には、通常のインキュベーション条件とすることにより不安定な物質であるＲＮＡは死菌中では減少し（生菌中では細胞内ではＲＮＡ量はほぼ一定である）、また、ＤＮＡの場合には、例えば特開２００８−６７７１８号公報に開示される、死菌および損傷菌のＤＮＡを酵素により選択的に分解させる条件下でインキュベーションすることにより、インキュベーションの時間の経過に伴って核酸の量を減少させることができる。 Furthermore, it is preferable that the incubation conditions described above are such that when the dead microorganism of the microorganism to be detected is present in the test sample, the amount of nucleic acid of the dead microorganism decreases as the culture time elapses. By incubating under such conditions, the change in the amount of nucleic acid between live and dead bacteria over the course of the incubation time is differentiated, and live and dead bacteria are processed by subsequent steps based on how the amount of nucleic acid changes. Can be determined. Many such conditions are known in the art. For example, in the case of RNA, RNA, which is an unstable substance, is reduced in dead cells by using normal incubation conditions (cells in live cells are reduced). In the case of DNA, for example, disclosed in Japanese Patent Application Laid-Open No. 2008-67718, under conditions where the DNA of dead and damaged bacteria is selectively degraded by an enzyme. Incubation can reduce the amount of nucleic acid over time.
本発明の方法の工程(b)では、工程(a)のインキュベーションの時間ｔ0およびｔ1（ｔ1＞ｔ0）における試料から核酸を抽出する。具体的な時間ｔ0およびｔ1は、検出対象の微生物の種類、インキュベーションの条件（温度、振とうの有無等）などを考慮して、本発明の方法を有効に実施できる程度に生菌が増殖するのに必要な時間を基に設定することができる。ｔ0は典型的にはインキュベーションの開始直後であり（ｔ0＝０）、（ｔ1−ｔ0）は例えば１日程度、好ましくは６〜１２時間程度、より好ましくは２〜６時間とすることができるが特に限定はされず、検出対象とする微生物の種類、使用する培地などを考慮して決定することができる。
また、抽出する核酸はＤＮＡまたはＲＮＡのいずれでもよく、またその抽出法は当業者によく知られている。ＤＮＡの場合には、proKなどの酵素や界面活性剤により溶菌したものからＤＮＡを抽出し、最終的にはフェノール/クロロホルム抽出、エタノール沈殿により精製することができる。また、ＲＮＡの場合には、AGPC法、NP-40法、セシウム超遠心法などにより抽出することができる。各種市販のキットを使用してもよく、ＤＮＡの場合は、例えばQIAGEN社製 DNeasy Blood & Tissue Kit、富士フイルム社製 自動核酸抽出装置 QuickGene-810 QuickGene SP Kit、TaKaRaBio社製 FastPure DNA Kit、MoBio社製 UltraClean Microbial DNA Kitなど、ＲＮＡの場合は、例えばQIAGEN社製 RNeasy Mini Kit、invitrogen社製 TRIzol Max Bacterial RNA Isolation Kit、TakaraBio社製 FastPure RNA Kitなどを使用することができる。
In step (b) of the method of the present invention, nucleic acids are extracted from the sample at the incubation times t 0 and t 1 (t 1 > t 0 ) of step (a). The specific times t 0 and t 1 are determined based on the type of microorganisms to be detected, incubation conditions (temperature, presence / absence of shaking, etc.), etc. It can be set based on the time required for growth. t 0 is typically immediately after the start of incubation (t 0 = 0), and (t 1 -t 0 ) is, for example, about 1 day, preferably about 6 to 12 hours, more preferably 2 to 6 hours. However, it is not particularly limited, and can be determined in consideration of the type of microorganism to be detected, the culture medium to be used, and the like.
The nucleic acid to be extracted may be either DNA or RNA, and the extraction method is well known to those skilled in the art. In the case of DNA, DNA can be extracted from those lysed by an enzyme such as proK or a surfactant, and finally purified by phenol / chloroform extraction and ethanol precipitation. In the case of RNA, it can be extracted by AGPC method, NP-40 method, cesium ultracentrifugation method and the like. Various commercially available kits may be used. In the case of DNA, for example, DNeasy Blood & Tissue Kit manufactured by QIAGEN, automated nucleic acid extraction device QuickGene-810 QuickGene SP Kit manufactured by Fuji Film, FastPure DNA Kit manufactured by TaKaRaBio, MoBio In the case of RNA such as UltraClean Microbial DNA Kit manufactured by QIAGEN, for example, RNeasy Mini Kit manufactured by QIAGEN, TRIzol Max Bacterial RNA Isolation Kit manufactured by invitrogen, FastPure RNA Kit manufactured by TakaraBio, etc. can be used.
本発明の方法の工程(c)では、時間ｔ0およびｔ1において抽出した前記それぞれの核酸を鋳型として、検出対象とする微生物に特異的な検出対象領域を含む核酸配列を増幅させ、それぞれ増幅核酸試料ａ0および増幅核酸試料ａ1を得る。検出対象とする微生物に特異的な検出対象領域としては、例えば16S-23S 遺伝子間スペーサー領域（internal transcribed spacer (ITS)）、16S rRNA、23S rRNA などの配列から、検出対象の各微生物に特異性の高い領域を選択することができる。このような検出対象領域を含む核酸配列の増幅はＰＣＲ法またはＲＴ−ＰＣＲ（Reverse Transcription PCR）法により行うことができる。
増幅に使用するプライマーは、検出対象とする微生物の配列に合わせて設計され、微生物毎に異なるプライマーセットを使用してもよいし、検出対象とする微生物群に特異的で微生物間で保存性の高い領域を２箇所選択し、その２つの領域の間の核酸配列を特異的に増幅し得るプライマーを設計して使用してもよい。例えば、16S rRNA 配列の保存性の高い部分にForwardプライマー、23S rRNA 配列の保存性の高い領域にReverseプライマーを設計し、この２つの領域に挟まれる16S-23S ITS領域（各微生物種に特異的な配列を含む）を増幅することができる（図１）。
好ましくは、数多くの微生物を検出するための簡便性を考慮して、検出対象とする複数の微生物種または菌株のそれぞれの種または菌株に特異的な検出対象領域を含む核酸配列を、前記核酸配列のいずれをも増幅し得る単一のプライマーセットにより増幅することが好ましい。プライマーの長さは、選択した配列の特異性等を考慮して当業者が適宜設定することが可能であり、例えば10〜50 mer、好ましくは15〜25 mer、例えば20 mer程度とすることができる。
In step (c) of the method of the present invention, a nucleic acid sequence containing a detection target region specific to the microorganism to be detected is amplified using the respective nucleic acids extracted at times t 0 and t 1 as templates, and each amplification A nucleic acid sample a 0 and an amplified nucleic acid sample a 1 are obtained. Specific detection target regions for the target microorganism include, for example, 16S-23S intergenic spacer region (ITS), 16S rRNA, 23S rRNA, and other specific sequences. Can be selected. Amplification of a nucleic acid sequence including such a detection target region can be performed by PCR or RT-PCR (Reverse Transcription PCR).
Primers used for amplification are designed according to the sequence of the microorganism to be detected, and different primer sets may be used for each microorganism, or they are specific to the microorganism group to be detected and are conserved among microorganisms. Two high regions may be selected and primers that can specifically amplify the nucleic acid sequence between the two regions may be designed and used. For example, a forward primer is designed for the highly conserved portion of the 16S rRNA sequence, and a reverse primer is designed for the highly conserved region of the 23S rRNA sequence, and the 16S-23S ITS region sandwiched between these two regions (specific to each microbial species) Can be amplified (Figure 1).
Preferably, taking into account the convenience for detecting a large number of microorganisms, a nucleic acid sequence comprising a detection target region specific to each species or strain of a plurality of microorganism species or strains to be detected, It is preferable to amplify with a single primer set capable of amplifying any of the above. The length of the primer can be appropriately set by those skilled in the art in consideration of the specificity of the selected sequence and the like, for example, 10 to 50 mer, preferably 15 to 25 mer, for example about 20 mer. it can.
本発明の方法の工程(d)では、増幅核酸試料ａ0および増幅核酸試料ａ1を、前記検出対象領域にハイブリダイズ可能なプローブが固定化された担体に接触させる。前記プローブは、検出対象とする各微生物に特異的な配列である前述の検出対象領域とハイブリダイズ可能な核酸の断片であり、例えば10〜50 mer、好ましくは10〜40 mer、典型的には20〜30 mer程度とすることができる。前記プローブが固定化された担体としては特に限定はされず、ビーズ、タイタープレート、マイクロアレイなどが挙げられるが、特に検出対象微生物の種類を多くする場合や、検出感度、再現性、自動化の容易さなどの要素を考慮してマイクロアレイであることが好ましい。担体の作製方法および増幅核酸試料とプローブとの接触のさせ方は公知であり、適切な条件は当業者が適宜決定することができる。増幅核酸試料ａ0およびａ1は、それぞれ別の担体（同一のプローブまたはプローブ群が固定化された担体）に接触させて次の工程(e)で核酸の量を比較してもよいし、増幅核酸試料ａ0およびａ1の混合物をそれぞれ別の蛍光標識でラベリングし、それらの混合物を１つの担体に接触させて、各蛍光標識のシグナルを別々に検出することにより核酸の量を比較してもよい。 In step (d) of the method of the present invention, the amplified nucleic acid sample a 0 and the amplified nucleic acid sample a 1 are brought into contact with a carrier on which a probe capable of hybridizing to the detection target region is immobilized. The probe is a nucleic acid fragment capable of hybridizing with the aforementioned detection target region, which is a sequence specific to each microorganism to be detected, for example, 10 to 50 mer, preferably 10 to 40 mer, typically It can be about 20-30 mer. The carrier on which the probe is immobilized is not particularly limited, and examples thereof include beads, titer plates, and microarrays. Particularly, when the number of microorganisms to be detected is increased, detection sensitivity, reproducibility, and ease of automation. A microarray is preferable in consideration of such factors as: The method for preparing the carrier and the method for bringing the amplified nucleic acid sample into contact with the probe are known, and appropriate conditions can be appropriately determined by those skilled in the art. The amplified nucleic acid samples a 0 and a 1 may be brought into contact with different carriers (carriers on which the same probe or probe group is immobilized), and the amounts of nucleic acids may be compared in the next step (e). Each mixture of amplified nucleic acid samples a 0 and a 1 is labeled with a separate fluorescent label, the mixture is contacted with one carrier, and the amount of nucleic acid is compared by detecting the signal of each fluorescent label separately. May be.
本発明の方法の工程(e)では、増幅核酸試料ａ0について前記担体に固定化されたプローブにハイブリダイズした核酸の量と、増幅核酸試料ａ1について前記担体に固定化されたプローブにハイブリダイズした核酸の量を比較する。この工程では、増幅核酸試料ａ0およびａ1について、工程(a)におけるインキュベーションに起因する核酸量の変化に基づいて被検試料中の微生物の生死を判定し、生菌を検出する。インキュベーション中には生菌は増殖し、その核酸量も増殖に伴って増加するのに対し、死菌はインキュベーションしても増殖しないため核酸量も増加しない。よって、増幅核酸試料ａ0でハイブリダイズした核酸の量と比較して増幅核酸試料ａ1でハイブリダイズした核酸の量が増えた場合に微生物が生菌であると判定される。また、検出対象とする微生物の死菌が被検試料中に存在する場合に前記死菌の核酸量が培養時間の経過とともに減少する条件下で被検試料をインキュベーションすることにより生菌と死菌の差をより確実に検出することが可能であり、前記同様、増幅核酸試料ａ0でハイブリダイズした核酸の量と比較して増幅核酸試料ａ1でハイブリダイズした核酸の量が同等もしくは増えた場合に微生物が生菌であると判定される（図２参照）。
検出対象とする微生物の死菌が被検試料中に存在する場合に前記死菌の核酸量が培養時間の経過とともに減少する条件は、核酸がＲＮＡである場合には、通常のインキュベーション条件とすることにより不安定な物質であるＲＮＡは死菌中で時間の経過と共に分解されて減少するため、一般にこの条件が満たされる。また、ＤＮＡの場合には、例えば特開２００８−６７７１８号公報に開示される、死菌および損傷菌のＤＮＡを酵素により選択的に分解させる条件下でインキュベーションすることにより、検出対象とする微生物の死菌が被検試料中に存在する場合に前記死菌の核酸量が培養時間の経過とともに減少するという条件を満たすことができる。この方法により生菌と死菌を区別することができるため、死菌の核酸が増幅されることにより見かけ上陽性と判定されてしまう偽陽性の検出を防止することができる。
核酸の検出およびその定量は使用する担体の種類等に応じて当技術分野に知られる方法を用いることができる。例えば担体としてマイクロアレイを使用した場合、工程(c)における核酸配列の増幅時にCy-3、Cy-5等の蛍光標識を導入することによって、プローブにハイブリダイズした核酸を蛍光シグナルとして検出および定量することができる。
In the step (e) of the method of the present invention, the amount of nucleic acid hybridized to the probe immobilized on the carrier for the amplified nucleic acid sample a 0 and the probe immobilized to the carrier for the amplified nucleic acid sample a 1 are hybridized. Compare the amount of soy nucleic acid. In this step, the amplified nucleic acid sample a 0 and a 1, to determine the life or death of the microorganisms in the test sample based on a change in the amount of nucleic acids resulting from incubation in step (a), the detecting live cells. During incubation, viable bacteria grow and the amount of nucleic acid increases with growth, whereas dead bacteria do not grow even when incubated, so the amount of nucleic acid does not increase. Therefore, when the amount of nucleic acid hybridized with the amplified nucleic acid sample a 1 is increased compared to the amount of nucleic acid hybridized with the amplified nucleic acid sample a 0 , it is determined that the microorganism is a live cell. In addition, when a dead microorganism of a microorganism to be detected is present in a test sample, the test sample is incubated under a condition in which the nucleic acid amount of the dead bacteria decreases with the passage of the culture time, whereby the live and dead bacteria are As described above, the amount of nucleic acid hybridized with the amplified nucleic acid sample a 1 was equal or increased as compared with the amount of nucleic acid hybridized with the amplified nucleic acid sample a 0 . In this case, it is determined that the microorganism is a living microbe (see FIG. 2).
When the dead microorganism of the microorganism to be detected is present in the test sample, the condition that the nucleic acid amount of the dead microorganism decreases with the passage of the culture time is the normal incubation condition when the nucleic acid is RNA. As a result, RNA, which is an unstable substance, is degraded and decreases over time in dead bacteria, so this condition is generally satisfied. In the case of DNA, for example, as disclosed in Japanese Patent Application Laid-Open No. 2008-67718, the microorganisms to be detected can be detected by incubation under conditions in which the DNA of dead and damaged bacteria is selectively degraded by an enzyme. When dead bacteria are present in the test sample, the condition that the nucleic acid amount of the dead bacteria decreases with the passage of the culture time can be satisfied. Since live bacteria and dead bacteria can be distinguished by this method, detection of false positives that are apparently determined to be positive when nucleic acids of dead bacteria are amplified can be prevented.
Nucleic acid detection and quantification can be carried out by methods known in the art depending on the type of carrier used. For example, when a microarray is used as a carrier, nucleic acid hybridized to the probe is detected and quantified as a fluorescent signal by introducing a fluorescent label such as Cy-3 or Cy-5 during the amplification of the nucleic acid sequence in step (c). be able to.
本発明の方法は、好ましくは核酸配列がハイブリダイズしたプローブの配列に基づいて微生物の種類を同定する工程をさらに含む。担体に固定化されたプローブは、検出対象とする各微生物の検出対象領域を検出するものであるため、担体の作製時に各プローブの由来微生物と担体との関係を特定してデータベース化しておくことにより、核酸の検出と同時にその微生物の種類を同定することができる（図３参照）。本発明の方法では、マイクロアレイを使う場合はプローブと微生物のアレイ上の位置の関係をデータベース化しておけば、核酸の検出の後にさらなる遺伝子解析を行うことなく、数多くの検出対象微生物の種類を瞬時に同定することができる。検出対象とする微生物の種類は、例えば５種類以上、好ましくは１０種類以上、より好ましくは３０種以上などにすることができるが、担体に固定化し得るプローブの数の範囲内であれば制限されない。 The method of the present invention preferably further comprises the step of identifying the type of microorganism based on the sequence of the probe to which the nucleic acid sequence is hybridized. Since the probe immobilized on the carrier detects the detection target region of each microorganism to be detected, the relation between the probe-derived microorganism and the carrier should be specified and created in a database when the carrier is prepared. Thus, the type of the microorganism can be identified simultaneously with the detection of the nucleic acid (see FIG. 3). In the method of the present invention, when a microarray is used, if the relationship between the positions of the probe and the microorganism on the array is stored in a database, a large number of types of microorganisms to be detected can be instantly detected without further gene analysis after nucleic acid detection. Can be identified. The number of microorganisms to be detected can be, for example, 5 or more, preferably 10 or more, more preferably 30 or more, but is not limited as long as it is within the number of probes that can be immobilized on a carrier. .
本発明の方法は、好ましくは工程(a)〜(e)の一部またはすべてが、自動で、かつ外部からの汚染がない環境下で行われる。このようにすることにより、被検試料中の生菌の検出を迅速、簡便かつ精密に行うことができる。 The method of the present invention is preferably carried out in an environment in which some or all of steps (a) to (e) are automatic and free from external contamination. By doing in this way, the detection of the living microbe in a test sample can be performed rapidly, simply and precisely.
以下、本発明のより具体的な実施態様の一例を示す。
食品サンプル25 gを汎用の標準培地（精製水１L当たり酵母エキス2.5g、トリペプト5g、ブドウ糖1g；pH 7.1〜7.5）225 ｍｌに懸濁し、機械的に破砕する。これを35℃の温度条件下でインキュベートし、インキュベート開始時および４時間後に一部をサンプリングする。
TaKaRaBio社製 FastPure DNA Kitを用い、その説明書に従って前記サンプリングした試料からＤＮＡを抽出する。抽出した前記ＤＮＡを鋳型として、以下の条件でＰＣＲを行い、ＤＮＡを増幅する。
25 g of a food sample is suspended in 225 ml of a general-purpose standard medium (2.5 g of yeast extract per liter of purified water, 5 g of tripept, 1 g of glucose; pH 7.1 to 7.5) and mechanically crushed. This is incubated under a temperature condition of 35 ° C., and a part is sampled at the start of incubation and after 4 hours.
Using FastPure DNA Kit manufactured by TaKaRaBio, DNA is extracted from the sampled sample according to the instructions. Using the extracted DNA as a template, PCR is performed under the following conditions to amplify the DNA.
プライマーとしては、６種類の微生物（Bacillus coagulans、Geobacillus stearothermophilus、Bacillus subtilis、Bacillus megaterium、Bacillus licheniformis、Paenibacillus polymyxa）の 16S rRNA 領域および 23S rRNA 領域の中から各微生物間で保存性の高い部分を基に設計され、16S rRNA 領域と 23S rRNA 領域との間にある16S-23S ITS領域を上記６種類の微生物について増幅することができることが確認されている以下のプライマーを、それぞれForward プライマーおよびReverse プライマーとして使用する。
・Forward プライマー（16S rRNA 領域）
ITS-FP-1：CCGCGGTGAATACGTTCC（配列番号：１）
・Reverse プライマー（23S rRNA 領域）
RP-1a：CTCCTAGTGCCAAGGCATCC（配列番号：２）
Primers are based on the 16S rRNA region and 23S rRNA region of 6 types of microorganisms ( Bacillus coagulans , Geobacillus stearothermophilus , Bacillus subtilis , Bacillus megaterium , Bacillus licheniformis , Paenibacillus polymyxa ). The following primers that have been designed and confirmed to be able to amplify the 16S-23S ITS region between the 16S rRNA region and the 23S rRNA region for the above six types of microorganisms are used as the Forward primer and the Reverse primer, respectively. To do.
・ Forward primer (16S rRNA region)
ITS-FP-1: CCGCGGTGAATACGTTCC (SEQ ID NO: 1)
・ Reverse primer (23S rRNA region)
RP-1a: CTCCTAGTGCCAAGGCATCC (SEQ ID NO: 2)
サーマルサイクラーとしてTOYOBO社製 LittleGeneを使用し、以下の温度条件により反応を行う。
微生物の検出対象領域を検出するための担体としてはマイクロアレイを使用し、前述の６種類の微生物（Bacillus coagulans、Geobacillus stearothermophilus、Bacillus subtilis、Bacillus megaterium、Bacillus licheniformis、Paenibacillus polymyxa）のそれぞれに特異的な16S-23S ITS領域（塩基数 20〜24 mer Tm値 53〜63℃）にハイブリダイズ可能なプローブを設計し、それらをマイクロアレイ上に固定化する。作製したマイクロアレイに対し、前記のＰＣＲにより増幅したＤＮＡ産物を、室温〜50℃で1〜16時間ハイブリダイズさせる。ハイブリダイズ溶液は、［PCR産物：ハイブリダイズバッファー（10×SSC/0.5% SDS） = 1：1］の溶液である（20×SSC：3M NaCl、0.3M クエン酸ナトリウム、SDS：ドデシル硫酸ナトリウム）。
次いで、核酸をハイブリダイズさせたマイクロアレイを、室温で、2×SSC/0.2% SDSで１回、2×SSCで２回洗浄する。
A microarray is used as a carrier for detecting the detection target region of microorganisms, and 16S specific to each of the above-mentioned six types of microorganisms ( Bacillus coagulans , Geobacillus stearothermophilus , Bacillus subtilis , Bacillus megaterium , Bacillus licheniformis , Paenibacillus polymyxa ). Probes that can hybridize to the -23S ITS region (base number: 20 to 24 mer Tm value: 53 to 63 ° C) are immobilized on a microarray. The DNA product amplified by the above PCR is hybridized to the prepared microarray at room temperature to 50 ° C. for 1 to 16 hours. The hybridization solution is a solution of [PCR product: hybridization buffer (10 × SSC / 0.5% SDS) = 1: 1] (20 × SSC: 3M NaCl, 0.3M sodium citrate, SDS: sodium dodecyl sulfate) .
The microarray hybridized with nucleic acid is then washed once with 2 × SSC / 0.2% SDS and twice with 2 × SSC at room temperature.
蛍光は、基盤の非スポット部をバックグラウンドとしてCy5の蛍光を検出する。
解析機により各蛍光強度算出し、インキュベーション開始時および４時間後における蛍光強度の増減算出して、インキュベーション４時間後に蛍光強度が増加した場合に、被検試料中におけるその微生物が生菌であったと判定する。同時に、シグナルが検出されたプローブの位置に基づいて微生物の種類を同定する。
For fluorescence, Cy5 fluorescence is detected using the non-spot portion of the substrate as a background.
Each fluorescence intensity is calculated by an analyzer, and the increase / decrease of the fluorescence intensity at the start of incubation and after 4 hours is calculated. When the fluorescence intensity increases after 4 hours of incubation, the microorganism in the test sample is live judge. At the same time, the type of microorganism is identified based on the position of the probe where the signal is detected.
Claims (5)
(b) 前記インキュベーションの時間ｔ0およびｔ1（ｔ1＞ｔ0）における前記試料から核酸を抽出する工程、
(c) 時間ｔ0およびｔ1において抽出した前記それぞれの核酸を鋳型として、検出対象とする微生物に特異的な検出対象領域を含む核酸配列を増幅させ、それぞれ増幅核酸試料ａ0および増幅核酸試料ａ1を得る工程、
(d) 増幅核酸試料ａ0および増幅核酸試料ａ1を、前記検出対象領域にハイブリダイズ可能なプローブが固定化された担体に接触させる工程、および
(e) 増幅核酸試料ａ0について前記担体に固定化されたプローブにハイブリダイズした核酸の量と、増幅核酸試料ａ1について前記担体に固定化されたプローブにハイブリダイズした核酸の量を比較する工程、
を含む、被検試料中の微生物の生菌を検出する方法。 (a) incubating the test sample under conditions that allow microorganisms that may be contained in the sample to grow;
(b) extracting nucleic acid from the sample at the incubation times t 0 and t 1 (t 1 > t 0 );
(c) Using each of the nucleic acids extracted at times t 0 and t 1 as a template, a nucleic acid sequence including a detection target region specific to the detection target microorganism is amplified, and an amplified nucleic acid sample a 0 and an amplified nucleic acid sample, respectively obtaining a 1 ,
(d) contacting the amplified nucleic acid sample a 0 and the amplified nucleic acid sample a 1 with a carrier on which a probe capable of hybridizing to the detection target region is immobilized; and
(e) Compare the amount of nucleic acid hybridized to the probe immobilized on the carrier for the amplified nucleic acid sample a 0 and the amount of nucleic acid hybridized to the probe immobilized to the carrier for the amplified nucleic acid sample a 1 Process,
A method for detecting viable microorganisms in a test sample.
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| WO2011129091A1 (en) * | 2010-04-14 | 2011-10-20 | 東洋製罐株式会社 | Primer set for pcr, reaction liquid for pcr, and method for detecting food poisoning bacteria |
| JP2012050362A (en) * | 2010-08-31 | 2012-03-15 | Toyo Seikan Kaisha Ltd | Primer set for multiplex pcr and method for detecting microorganism using the same |
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| WO2002052034A1 (en) * | 2000-12-26 | 2002-07-04 | Joji Oshima | Bioscopy method and method of nucleic acid amplification |
| JP2003517843A (en) * | 1999-12-15 | 2003-06-03 | コナグラ・グロサリー・プロダクツ・カンパニーズ | Multi-food testing and characterization |
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| JP2003517843A (en) * | 1999-12-15 | 2003-06-03 | コナグラ・グロサリー・プロダクツ・カンパニーズ | Multi-food testing and characterization |
| WO2002052034A1 (en) * | 2000-12-26 | 2002-07-04 | Joji Oshima | Bioscopy method and method of nucleic acid amplification |
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| WO2011129091A1 (en) * | 2010-04-14 | 2011-10-20 | 東洋製罐株式会社 | Primer set for pcr, reaction liquid for pcr, and method for detecting food poisoning bacteria |
| CN102844433A (en) * | 2010-04-14 | 2012-12-26 | 东洋制罐株式会社 | Primer set for pcr, reaction liquid for pcr, and method for detecting food poisoning bacteria |
| CN102844433B (en) * | 2010-04-14 | 2015-08-05 | 东洋制罐株式会社 | The detection method of PCR primer pair, PCR reaction solution and food poisoning bacteria |
| JP2012050362A (en) * | 2010-08-31 | 2012-03-15 | Toyo Seikan Kaisha Ltd | Primer set for multiplex pcr and method for detecting microorganism using the same |
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