JP2015039361A - Analysis method for saliva bacterial flora - Google Patents
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Abstract
Description
本発明は唾液の細菌叢の解析方法に関する。 The present invention relates to a method for analyzing the bacterial flora of saliva.
齲蝕の発症に口腔内の微生物が決定的な原因子として働くことは周知の事実である。一方、従来の齲蝕病因論では、ミュータンス連鎖球菌が主要な齲蝕原性細菌とされるが、これだけで成人の齲蝕病態を十分に説明することができなかった。
デンタルプラークはバイオフィルムの成熟に伴い、細菌の種類ならびに各細菌の割合が変化することでその病原性が変化すると考えられる。これまでデンタルプラークを構成する細菌叢の経時的変化については１９６７年にＲｉｔｚ ＨＬが報告した結果（非特許文献１）に報告がある。しかし、この報告のみでは、複雑な細菌叢を背景とする齲蝕原性バイオフィルムの詳細な構成を解明するには多くの課題を残している。
It is a well-known fact that microorganisms in the oral cavity act as a critical causative factor in the development of caries. On the other hand, in the conventional caries etiology, Streptococcus mutans is the main cariogenic bacterium, but this alone could not fully explain the caries pathology of adults.
Dental plaque is thought to change its pathogenicity as the type of bacteria and the proportion of each bacteria change as the biofilm matures. The change over time of the bacterial flora constituting dental plaque has been reported in the results reported by Ritz HL in 1967 (Non-patent Document 1). However, this report alone leaves many challenges to elucidate the detailed composition of cariogenic biofilms against the background of complex bacterial flora.
一方、近年の分子遺伝工学手法の発達により、細菌群集の解析を培養法に頼ることなく実施できるようになり、複雑な細菌叢の解析に新しい取り組みが進められるようになってきた。発明者らは以前、新しい細菌叢分析法として独自に開発した改良Ｔｅｒｍｉｎａｌ−Ｒｅｓｔｒｉｃｔｉｏｎ Ｆｒａｇｍｅｎｔ Ｌｅｎｇｔｈ Ｐｏｌｙｍｏｒｐｈｉｓｍ（Ｔ−ＲＦＬＰ）法を用いて、バイオフィルムとして形成されるデンタルプラークの成熟過程を経日的に追跡することに成功し、興味ある知見を得た（特許文献１）。
しかし、デンタルプラークの解析は、プラークの回収が煩雑であり、簡易検査には不向きであった。本発明では、簡易に回収が可能な、唾液を用いた口腔内の細菌叢の解析方法、あるいは、診断を補助するためのデータ収集方法を見出し、さらに、これらの方法により、齲蝕のなりにくさを判断する方法を見出した。
On the other hand, with the recent development of molecular genetic engineering techniques, it has become possible to carry out bacterial community analysis without relying on culture methods, and new efforts have been made to analyze complex bacterial flora. Inventors previously tracked the maturation process of dental plaque formed as a biofilm using the improved Terminal-Restriction Fragment Length Polymorphism (T-RFLP) method, which was originally developed as a new bacterial flora analysis method. We succeeded in doing so and gained interesting knowledge (Patent Document 1).
However, the analysis of dental plaque is not suitable for simple inspection because the plaque recovery is complicated. In the present invention, a method for analyzing the bacterial flora in the oral cavity using saliva, which can be easily collected, or a data collection method for assisting diagnosis, and further, these methods are less likely to cause caries. I found a way to judge.
唾液の菌叢中のＰｏｒｐｈｙｒｏｍｏｎａｓ，ＦｕｓｏｂａｃｔｅｒｉｕｍまたはＧｅｍｅｌｌａの存在割合を求めることを特徴とする、齲蝕のなりにくさの診断を補助するためのデータ収集方法あるいは解析方法を提供する。 Provided is a data collection method or analysis method for assisting diagnosis of difficulty in caries, characterized by determining the abundance ratio of Porphyromonas, Fusobacterium or Gemella in the saliva flora.
唾液の細菌叢の詳細な解析とそれに基づく齲蝕のなりにくさの判断を課題とする。 The detailed analysis of salivary bacterial flora and the determination of the difficulty of caries based on the analysis.
本発明は唾液の菌叢中のＰｏｒｐｈｙｒｏｍｏｎａｓ，ＦｕｓｏｂａｃｔｅｒｉｕｍまたはＧｅｍｅｌｌａの存在割合を求めることを特徴とする、齲蝕のなりにくさの診断を補助するためのデータ収集方法または解析方法を提供する。また、さらには、本発明は、菌叢中のＰｏｒｐｈｙｒｏｍｏｎａｓの割合が３．５％以上であれば齲蝕になりにくいと判断することを特徴とする齲蝕のなりにくさの診断を補助するためのデータ収集方法あるいは解析方法を提供する。上記齲蝕のなりにくさの診断を補助するためのデータ収集方法あるいは解析方法は菌叢中のＰｏｒｐｈｙｒｏｍｏｎａｓの割合が３．５％以上であれば齲蝕になりにくいと判断することを特徴とすることができる。また、齲蝕のなりにくさの診断を補助するためのデータ収集方法あるいは解析方法は、被験者から唾液を採取し、採取した唾液中のＤＮＡを抽出し、抽出されたＤＮＡ中の１６Ｓ ｒＲＮＡ遺伝子を増幅し、増幅された遺伝子の１０００リード以上あたりのＯＴＵ数の配列の解析を行うことを特徴とすることができる。また、齲蝕のなりにくさの診断を補助するためのデータ収集方法あるいは解析方法は、さらに１０００リードあたりＯＴＵ数が２７０以上であれば齲蝕傾向が低いと判断することを特徴とすることができる。
また、本発明は、上記に記載の齲蝕のなりにくさの診断を補助するためのデータ収集方法あるいは解析方法に用いられるキットを提供する。キットは、Ｐｏｒｐｈｙｒｏｍｏｎａｓ，ＦｕｓｏｂａｃｔｅｒｉｕｍまたはＧｅｍｅｌｌａに対する抗体を含むことを特徴とすることができる。
The present invention provides a data collection method or analysis method for assisting diagnosis of difficulty in caries, characterized by determining the presence ratio of Porphyromonas, Fusobacterium or Gemella in the saliva flora. Furthermore, the present invention provides data for assisting diagnosis of difficulty in caries, characterized in that it is determined that caries are unlikely to be caries if the proportion of Porphyromonas in the flora is 3.5% or more. Provide collection or analysis methods. The data collection method or analysis method for assisting diagnosis of difficulty in caries is characterized in that it is determined that caries are unlikely to be caries if the proportion of Porphyromonas in the flora is 3.5% or more. it can. In addition, a data collection method or analysis method for assisting diagnosis of difficulty in caries is to collect saliva from a subject, extract DNA in the collected saliva, and amplify 16S rRNA gene in the extracted DNA. Then, it is possible to analyze the sequence of the number of OTUs per 1000 reads or more of the amplified gene. Further, the data collection method or analysis method for assisting diagnosis of difficulty in caries can be characterized in that if the number of OTUs per 1000 leads is 270 or more, the caries tendency is low.
The present invention also provides a kit for use in the data collection method or analysis method for assisting diagnosis of the difficulty of caries as described above. The kit may be characterized in that it comprises an antibody against Porphyromonas, Fusobacterium or Gemella.
また、本発明は、被験者から唾液を採取し、採取した唾液中のＤＮＡを抽出し、抽出されたＤＮＡ中の１６Ｓ ｒＲＮＡ遺伝子を増幅し、増幅された遺伝子の配列の解析を行うことを特徴とする微生物の解析方法を提供する。
この方法においては、被験者から唾液を採取し、採取した唾液中のＤＮＡを抽出し、抽出されたＤＮＡ中の１６Ｓ ｒＲＮＡ遺伝子を増幅し、増幅された遺伝子の配列の解析を行うことを特徴とする、齲蝕になりにくさを診断することができる。この方法においては、さらに１０００リードあたりのＯＴＵ数を求めることを特徴とすることができ、さらには、１０００リードあたりＯＴＵ数が２７０以上であれば齲蝕になりにくいと判断することができる。この方法においては、さらに菌叢中の菌叢中のＰｏｒｐｈｙｒｏｍｏｎａｓ，ＦｕｓｏｂａｃｔｅｒｉｕｍまたはＧｅｍｅｌｌａの割合を求めることを特徴とすることができ、菌叢中のＰｏｒｐｈｙｒｏｍｏｎａｓの割合が３．５％以上であれば齲蝕になりにくいと判断することができる。
なお、齲蝕になりにくいとは、齲蝕の経験がない、または、今後齲蝕になりにくい傾向であることを意味し、齲蝕になりやすいとは、齲蝕の経験がこれまでにある、または、今後、齲蝕になりやすい傾向であることを意味する。
Further, the present invention is characterized in that saliva is collected from a subject, DNA in the collected saliva is extracted, 16S rRNA gene in the extracted DNA is amplified, and the sequence of the amplified gene is analyzed. Provided is a method for analyzing microorganisms.
In this method, saliva is collected from a subject, DNA in the collected saliva is extracted, 16S rRNA gene in the extracted DNA is amplified, and the sequence of the amplified gene is analyzed. Can diagnose the difficulty of getting caries. In this method, the number of OTUs per 1000 leads can be further obtained, and further, if the number of OTUs per 1000 leads is 270 or more, it can be determined that caries are not likely to be caries. In this method, the ratio of Porphyromonas, Fusobacterium or Gemella in the flora of the flora can be further determined, and caries can be caries if the ratio of Porphyromonas in the flora is 3.5% or more. It can be judged that it is difficult to become.
In addition, being hard to become caries means that there is no experience of caries, or that it tends to be hard to become caries in the future, and that it is likely to become caries means that there has been experience of caries so far, It means that it tends to be caries.
本発明においては、１６Ｓ ｒＲＮＡ遺伝子を増幅する際に、マイクロリアクター内で増幅し、遺伝子の配列の解析は、パイロシーケンス法により行うことができる。また、１６Ｓ ｒＲＮＡ遺伝子を増幅する際に、検体に特有のタグ配列を付加したプライマーを使用することができる。また、本発明においては、被験者にパラフィンワックスを咀嚼させることにより唾液分泌を促すことによって、唾液を採取することができる。 In the present invention, when a 16S rRNA gene is amplified, it is amplified in a microreactor, and the gene sequence can be analyzed by a pyrosequencing method. In addition, when a 16S rRNA gene is amplified, a primer to which a tag sequence peculiar to a specimen is added can be used. In the present invention, saliva can be collected by encouraging saliva secretion by chewing paraffin wax by the subject.
本発明の方法によれば、簡易的に採取が可能な唾液に基づいて、細菌叢の詳細な解析が可能となった。マイクロリアクターを用いることで大量の遺伝子情報を得られる。パイロシーケンス法により行うことで、比較的安価に高速に大量の解析を行うことができる。また、検体に特有のタグ配列を付加したプライマーを使用することにより、得られた結果の情報処理が容易となる。 According to the method of the present invention, detailed analysis of bacterial flora is possible based on saliva that can be collected easily. A large amount of genetic information can be obtained by using a microreactor. By using the pyro sequence method, a large amount of analysis can be performed at a relatively low cost and at a high speed. Further, by using a primer to which a tag sequence peculiar to a specimen is used, information processing of the obtained result is facilitated.
さらには、実施例に示すように、唾液の細菌叢の詳細な解析により、齲蝕経験群と齲蝕非経験群とで明確な差のある解析結果を得ることができる。すなわち、本発明の方法により、齲蝕になりにくさの診断、あるいは、診断を補助するデータ収集が可能となる。 Furthermore, as shown in the Examples, detailed analysis of the salivary bacterial flora can provide analysis results with a clear difference between the caries-experienced group and the caries-inexperienced group. That is, according to the method of the present invention, it is possible to diagnose the difficulty of caries or to collect data to assist the diagnosis.
本発明は、被験者から唾液を採取し、採取した唾液中のＤＮＡを抽出し、抽出されたＤＮＡ中の１６Ｓ ｒＲＮＡ遺伝子を増幅し、増幅された遺伝子の配列の解析を行うことを特徴とする唾液の細菌叢の解析方法を提供する。
また、本発明は被験者から唾液を採取し、採取した唾液中のＤＮＡを抽出し、抽出されたＤＮＡ中の１６Ｓ ｒＲＮＡ遺伝子を増幅し、増幅された遺伝子の配列の解析を行うことを特徴とする、齲蝕のなりにくさの診断方法を提供する。
さらに、本発明は被験者から唾液を採取し、採取した唾液中のＤＮＡを抽出し、抽出されたＤＮＡ中の１６Ｓ ｒＲＮＡ遺伝子を増幅し、増幅された遺伝子の配列の解析を行うことを特徴とする、齲蝕のなりにくさの診断を補助するためのデータ収集方法を提供する。
The present invention is characterized by collecting saliva from a subject, extracting DNA in the collected saliva, amplifying a 16S rRNA gene in the extracted DNA, and analyzing the sequence of the amplified gene A method for analyzing bacterial flora is provided.
The present invention is also characterized in that saliva is collected from a subject, DNA in the collected saliva is extracted, 16S rRNA gene in the extracted DNA is amplified, and the sequence of the amplified gene is analyzed. To provide a method for diagnosing the difficulty of caries.
Furthermore, the present invention is characterized in that saliva is collected from a subject, DNA in the collected saliva is extracted, 16S rRNA gene in the extracted DNA is amplified, and the sequence of the amplified gene is analyzed. To provide a data collection method for assisting diagnosis of difficulty in caries.
１６Ｓ ｒＲＮＡとはリボソームのサブユニットを構成するＲＮＡである。リボソームは配列の保存性が高く、生物種の比較の際の目安となりやすい。中でも１６Ｓ ｒＲＮＡは適度な長さを持っており、生物種の決定に好都合に用いられる。
細菌叢とは、特定の環境中に存在する細菌の集合をいう。本明細書において、細菌とは、微生物のことを指し、真正細菌のみでなく、古細菌、真核生物、菌類、粘菌、ウィルスなどを含む。
16S rRNA is RNA constituting a subunit of ribosome. Ribosomes are highly conserved in sequences, and can easily be used as a guide when comparing species. Among these, 16S rRNA has an appropriate length, and is conveniently used for the determination of species.
The bacterial flora refers to a collection of bacteria present in a specific environment. In this specification, bacteria refer to microorganisms and include not only eubacteria but also archaea, eukaryotes, fungi, slime molds, viruses and the like.
齲蝕になりにくさ、あるいはなりやすさとは、個人が齲蝕になる傾向を指す。同じ環境であっても、個人により、齲蝕になる場合と、ならない場合がある。一般的に、齲蝕経験のない者、あるいは少ない者は、齲蝕になりにくく、齲蝕経験の多い者は、齲蝕になりやすいと考えられる。
本実施例では、齲蝕経験群と齲蝕非経験群とで細菌の構成に差が見られることが分かった。被験者から採取して解析した細菌の構成が、齲蝕経験群の細菌構成に一致する、あるいは、類似すれば、齲蝕になりやすいと診断でき、齲蝕非経験群の細菌構成に一致する、あるいは類似すれば、齲蝕になりにくいと判断することができる。
The difficulty of becoming caries or the likelihood of being caries refers to the tendency of individuals to become caries. Even in the same environment, individuals may or may not become caries. In general, those who have little or no caries experience are less likely to get caries, and those who have a lot of caries experience are more likely to get caries.
In this example, it was found that there was a difference in the bacterial composition between the caries experience group and the caries non-experience group. If the bacterial composition collected and analyzed from the subject matches or resembles the bacterial composition of the caries-experienced group, it can be diagnosed that it is susceptible to caries, and matches or resembles the bacterial composition of the caries-inexperienced group. If it is, it can be judged that it is hard to become a caries.
本発明において、１６Ｓ ｒＲＮＡ遺伝子を増幅する際には、マイクロリアクター内で増幅されることができる。
マイクロリアクターとは、微小なサイズの反応容器のことであり、より具体的にはエマルジョンを挙げることができる。近年、大量遺伝子解析の際、あらかじめアダプターを付加された遺伝子を、アダプターを介してビーズに結合して、エマルジョン内で増幅することで、ビーズ上で、クローナルに遺伝子を増幅する技術が用いられている。
In the present invention, when a 16S rRNA gene is amplified, it can be amplified in a microreactor.
A microreactor is a reaction vessel of a minute size, and more specifically, an emulsion can be mentioned. In recent years, a technology that amplifies genes clonally on beads by attaching genes to adapters via adapters and amplifying them in emulsion has been used in large-scale gene analysis. Yes.
遺伝子の配列の解析は、パイロシーケンス法によることができる。パイロシーケンス法とは、遺伝子配列の解析の方法で、対象となるＤＮＡを鋳型として複製を行う際に、取り込こまれたヌクレオチドから放出されるピロリン酸を検出することを特徴とする方法である。ピロリン酸の検出には、ＡＴＰを介し、ルシフェリンの発光に基づくことが多い。たとえば、デオキシリボヌクレオチドを一種類ずつ加え、その際に、どの程度発光が起こるかを検出することで、どの遺伝子が取り込まれたことが分かり、遺伝子配列を決定することができる。 The gene sequence can be analyzed by a pyrosequencing method. The pyrosequencing method is a method for analyzing gene sequences, and is characterized by detecting pyrophosphate released from incorporated nucleotides when replication is performed using a target DNA as a template. . The detection of pyrophosphate is often based on luminescence of luciferin via ATP. For example, by adding deoxyribonucleotides one by one and detecting how much luminescence occurs at that time, it can be determined which gene has been incorporated and the gene sequence can be determined.
また、１６Ｓ ｒＲＮＡ遺伝子を増幅する際に検体に特有のタグ配列を付加したプライマーを使用することができる。タグ配列とは、遺伝子配列の解析の際に、試料がどの検体に由来するか知るための手がかりとすべく、人為的に挿入される配列のことである。 In addition, when a 16S rRNA gene is amplified, a primer to which a tag sequence peculiar to a specimen is added can be used. The tag sequence is a sequence that is artificially inserted in order to know which specimen the sample is derived from when analyzing the gene sequence.
本発明においては、唾液は被験者にパラフィンワックスを咀嚼させることにより唾液分泌を促すし、その後唾液を採取することができる。 In the present invention, saliva promotes saliva secretion by allowing a subject to chew paraffin wax, and then saliva can be collected.
また、本発明は、上記の細菌叢の解析方法あるいは、齲蝕になりにくさの診断を補助するためのデータ収集方法に用いるための、キットを提供する。
キットは、それぞれの方法を実施するために用いられる、各種試薬、バッファー、酵素、オリゴＤＮＡのほか、唾液を採取するための試験管などの道具、あるいは、唾液採取前に唾液の分泌を促すための咀嚼用のパラフィンワックス、チューインガム等を含むことができる。
In addition, the present invention provides a kit for use in the above-described method for analyzing bacterial flora or a data collection method for assisting diagnosis of difficulty in caries.
In addition to various reagents, buffers, enzymes, oligo DNA, tools such as test tubes for collecting saliva, and kits for promoting saliva secretion before collecting saliva. Paraffin wax for chewing, chewing gum and the like can be included.
以下、実施例により本発明をより詳細に説明する。なお、概略については、図１に図示されている。 Hereinafter, the present invention will be described in more detail with reference to examples. The outline is shown in FIG.
１．被験者
歯周組織が健康な２０歳から２８歳の１９名を被験者とし、齲蝕の経験の有無に基づき、被験者らを、齲蝕非経験群（９名：男性７名、女性２名）と齲蝕経験群（１０名：男性６名、女性４名）に分類した。齲蝕非経験群の９名については、平均年齢が２３．２±２．０歳、実験時点における歯数は２９．４±２．１本、齲蝕歯数、及び治療歯（齲蝕であったが既に治療された歯）数は０本、唾液の流量は１．４±０．６ｍｌ／分、唾液の緩衝能は中等度が２名、高度が７名であった。齲蝕非経験群の１０名については、平均年齢が２４．１±２．４歳、実験時点における歯数は２９．２±２．３本、齲蝕歯数は０．６±１．１本、治療歯数は１１．９±３．０本、唾液の流量は１．２±０．５ｍｌ／分、唾液の緩衝能は中等度が２名、高度が８名であった。
1. Subjects 19 subjects from 20 to 28 years old with healthy periodontal tissues were subjects, and based on the presence or absence of caries experience, subjects were caries-inexperienced group (9: 7 men, 2 women) and caries experience It was classified into groups (10 people: 6 men, 4 women). Nine caries inexperienced groups had an average age of 23.2 ± 2.0 years, 29.4 ± 2.1 teeth at the time of the experiment, the number of carious teeth, and the treated teeth (caries The number of teeth already treated was 0, the saliva flow rate was 1.4 ± 0.6 ml / min, and the buffer capacity of saliva was 2 for moderate and 7 for altitude. For the 10 people in the caries-inexperienced group, the average age was 24.1 ± 2.4 years, the number of teeth at the time of the experiment was 29.2 ± 2.3, the number of caries teeth was 0.6 ± 1.1, The number of treated teeth was 11.9 ± 3.0, the saliva flow rate was 1.2 ± 0.5 ml / min, and the buffer capacity of saliva was 2 for moderate and 8 for altitude.
２．唾液からのＤＮＡの抽出
被験者らは、パラフィンワックスを5分間、咀嚼し、咀嚼後の被験者らの唾液をそれぞれ試験管に採取した。
サンプル中に含まれるＤＮＡの抽出は、非特許文献２の方法を一部改良して行った。唾液を採取したチューブの中に０．３ｇのｚｉｒｃｏｎｉａ−ｓｉｌｉｃａ ｂｅａｄｓ（直径０．１ｍＭ：Ｂｉｏｓｐｅｃ Ｐｒｏｄｕｃｔｓ，ＵＳＡ）と１個のｔｕｎｇｓｔｅｎ−ｃａｒｂｉｄｅ ｂｅａｄ（直径３ｍＭ：Ｑｉａｇｅｎ，Ｇｅｒｍａｎｙ）を加えて９０℃で１０分間加温した後、Ｄｉｓｒｕｐｔｏｒ Ｇｅｎｉｅ（Ｓｃｉｅｎｔｉｆｉｃ Ｉｎｄｕｓｔｒｉｅｓ，Ｉｎｃ．，ＵＳＡ）を用いて菌体を震盪、破砕し、２００μｌの１％ ＳＤＳ溶液を加えて、７０℃で１０分間加温した。さらに、蛋白質成分を除去するため、フェノール（ｖ／ｖ）による抽出を１回、フェノール・クロロホルム・イソアミルアルコール（２５：２４：１、ｖ／ｖ）混合溶液による抽出を１回行った後、エタノール沈殿処理を行い、生じた沈殿物を５０μｌのＴＥ溶液（１ｍＭ ＥＤＴＡ を含む１０ｍＭトリス塩酸緩衝液 ； ｐＨ８．０）に溶解し、ＤＮＡ試料として分析時まで−３０℃で凍結保存した。
2. Extraction of DNA from saliva The subjects chewed paraffin wax for 5 minutes, and collected the saliva of the subjects after chewing into test tubes.
Extraction of DNA contained in the sample was performed by partially improving the method of Non-Patent Document 2. 0.3 g of zirconia-silica beads (diameter 0.1 mM: Biospec Products, USA) and one tungsten-carbide bead (diameter 3 mM: Qiagen, Germany) were added at 90 ° C. for 10 times. After warming for 5 minutes, the cells were shaken and crushed using Distortor Genie (Scientific Industries, Inc., USA), 200 μl of 1% SDS solution was added, and the mixture was heated at 70 ° C. for 10 minutes. Furthermore, in order to remove protein components, extraction with phenol (v / v) is performed once, and extraction with a mixed solution of phenol, chloroform and isoamyl alcohol (25: 24: 1, v / v) is performed once, and then ethanol is added. Precipitation treatment was carried out, and the resulting precipitate was dissolved in 50 μl of TE solution (10 mM Tris-HCl buffer containing 1 mM EDTA; pH 8.0) and stored frozen at −30 ° C. as a DNA sample until analysis.
３．４５４ Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ ｇｅｎｏｍｅ ｓｅｑｕｅｎｃｅｒ ＦＬＸ ｉｎｓｔｒｕｍｅｎｔによる遺伝子情報の取得
抽出ＤＮＡ試料から１６Ｓ ｒＲＮＡ遺伝子を増幅した。この際、アダプター配列と、検体ごとに人為的に取り決めた遺伝子配列（タグ）、が含まれるように増幅した。
すなわち、５’側にアダプター配列 Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ ａｄａｐｔｏｒ Ａと６塩基のタグ配列（配列２１：ＣＣＡ ＴＣＴ ＣＡＴ ＣＣＣ ＴＧＣ ＧＴＧ ＴＣＴ ＣＣＧ ＡＣＴ ＣＡＧ ＮＮＮ ＮＮＮ）を付与した８Ｆ（配列２２：ＡＧＡ ＧＴＴ ＴＧＡ ＴＹＭ ＴＧＧ ＣＴＣ ＡＧ）をフォワードプライマーとして、アダプター配列 Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ ａｄａｐｔｏｒ Ｂ（配列２３：ＣＣＴ ＡＴＣ ＣＣＣ ＴＧＴ ＧＴＧ ＣＣＴ ＴＧＧ ＣＡＧ ＴＣＴ ＣＡＧ）を付与した３３８Ｒ（配列２４：ＧＧＡ ＣＴＡ ＣＣＲ ＧＧＧ ＴＡＴ ＣＴＡ Ａ）をリバースプライマーとしてＰＣＲ法により行った。用いたプライマーの配列を表１に示す。
3.454 Acquisition of gene information by Life Sciences genome sequencer FLX instrument The 16S rRNA gene was amplified from the extracted DNA sample. At this time, the amplification was performed so that the adapter sequence and the gene sequence (tag) artificially determined for each specimen were included.
That is, 8F (Sequence 22: AGA GTT TGA TYM TGT GTG TGT G ) With the adapter sequence Life Sciences adapter B (sequence 23: CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG) as a reverse primer using PCR as a reverse primer went. The primer sequences used are shown in Table 1.
ＰＣＲ反応にはＫＯＤ ＤＮＡポリメラーゼ（東洋紡績株式会社）を用いた。１μｌの鋳型ＤＮＡ（１００−５００ｎｇ／μｌになるよう希釈したもの）に５μｌのＫＯＤ ＤＮＡポリメラーゼ１０×ＰＣＲ ｂｕｆｆｅｒ（６０ｍＭ 硫酸アンモニウム、１００ｍＭ 塩化カルシウム、１％Ｔｒｉｔｏｎ Ｘ−１００、１００μｇウシ血清アルブミンを含む１．２Ｍトリス塩酸緩衝液；ｐＨ８．０）、５μｌの２ｍＭ ｄＮＴＰｓ、２μｌの２５ｍＭ 塩化マグネシウム、各０．５μｌの１μＭ両プライマー、１μｌのＫＯＤ ＤＮＡポリメラーゼ（２．５Ｕ／μｌ）を加えた後、滅菌蒸留水を加えて総量を５０μｌとしてＰＣＲ反応を行った。ＰＣＲ反応にはＢｉｏｍｅｔｒａ Ｔ３ ｔｈｅｒｍｏｃｙｃｌｅｒ（Ｂｉｏｍｅｔｒａ，Ｇｅｒｍａｎｙ）を用いた。反応条件は９８℃ １５秒、６０℃ ２秒、７２℃ ３０秒で３０サイクルの反応を行った。得られたＰＣＲ産物は、泳動用ゲルすなわち２％（ｗｔ／ｖｏｌ）のアガロースを含む１×ＴＡＥを用いて、アガロース電気泳動を行い、目的のバンド出現部位を切り出し、Ｗｉｚａｒｄ ＳＶ Ｇｅｌ ａｎｄ ＰＣＲ Ｃｌｅａｎ−Ｕｐ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ，ＵＳＡ）を用いて精製し、未反応プライマー、プライマーダイマー、その他非特異的増幅断片の除去を行った。 KOD DNA polymerase (Toyobo Co., Ltd.) was used for the PCR reaction. 1. 1 μl of template DNA (diluted to 100-500 ng / μl) containing 5 μl of KOD DNA polymerase 10 × PCR buffer (60 mM ammonium sulfate, 100 mM calcium chloride, 1% Triton X-100, 100 μg bovine serum albumin) 2M Tris-HCl buffer; pH 8.0) 5 μl 2 mM dNTPs, 2 μl 25 mM magnesium chloride, 0.5 μl each 1 μM both primers, 1 μl KOD DNA polymerase (2.5 U / μl) and then sterile distillation PCR was performed by adding water to make the total volume 50 μl. Biometra T3 thermocycler (Biometra, Germany) was used for the PCR reaction. The reaction conditions were 98 ° C. for 15 seconds, 60 ° C. for 2 seconds, and 72 ° C. for 30 seconds for 30 cycles. The obtained PCR product was subjected to agarose electrophoresis using 1 × TAE containing 2% (wt / vol) agarose for electrophoresis, the target band appearance site was excised, and Wizard SV Gel and PCR Clean- Purification was carried out using Up System (Promega, USA), and unreacted primers, primer dimers, and other non-specific amplified fragments were removed.
精製した１６Ｓ ｒＲＮＡ遺伝子増幅断片を含む各溶液はＮａｎｏＤｒｏｐ ｓｐｅｃｔｒｏｐｈｏｔｏｍｅｔｅｒ（ＮａｎｏＤｒｏｐ Ｔｅｃｈｎｏｌｏｇｉｅｓ Ｗｉｌｍｉｎｇｔｏｎ，ＤＥ）を用いＤＮＡ濃度を測定し、各検体について等濃度（５０ｎｇ／μｌ）になるように調整したのち混合した。混合検体は北海道システム・サイエンス株式会社に受託し、４５４ Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ ｇｅｎｏｍｅ ｓｅｑｕｅｎｃｅｒ ＦＬＸ ｉｎｓｔｒｕｍｅｎｔ（Ｒｏｃｈｅ，Ｂａｓｅｌ，Ｓｗｉｔｚｅｒｌａｎｄ）による塩基配列の解読を行った。
すなわち、上記で得られたアダプターが付加されたＤＮＡは、一本鎖にした後、アダプターを介してビーズに結合した。ビーズを油水エマルジョンの中に包み込み、ビーズ１つとＤＮＡフラグメント１つを持つマイクロリアクターを形成し、リアクター内にてＤＮＡ断片を増幅した。増幅反応後ＤＮＡフラグメントがビーズに結合している状態で油水エマルジョンを破壊した。
Each solution containing the purified 16S rRNA gene amplified fragment was measured for DNA concentration using NanoDrop Spectrophotometer (NanoDrop Technologies Wilmington, DE), adjusted to have an equal concentration (50 ng / μl) for each sample, and then mixed. The mixed sample was entrusted to Hokkaido System Science Co., Ltd., and the base sequence was deciphered using 454 Life Sciences gene sequencer FLX instrument (Roche, Basel, Switzerland).
That is, the DNA to which the adapter obtained above was added was made into a single strand and then bound to the bead through the adapter. The beads were wrapped in an oil-water emulsion to form a microreactor with one bead and one DNA fragment, and the DNA fragment was amplified in the reactor. After the amplification reaction, the oil-water emulsion was broken with the DNA fragments bound to the beads.
以上によって、ＤＮＡはビーズごとにクローナルに増幅された。これらのビーズを濃縮し、配列解析をするためのピコタイタープレート上に添加した。ピコタイタープレートを装置にセットし、それぞれの配列をパイロシークエンス法により解読した。すなわち、ポリメラーゼによりテンプレートに相補的な塩基が取り込まれた際に放出されるピロリン酸を、ルシフェリンの発光に基づき、ＣＣＤカメラで検出することにより、ＤＮＡの配列を解読した。 Thus, DNA was clonally amplified for each bead. These beads were concentrated and added onto a picotiter plate for sequence analysis. A picotiter plate was set in the apparatus, and each sequence was decoded by the pyrosequencing method. That is, pyrophosphoric acid released when a complementary base was incorporated into the template by the polymerase was detected by a CCD camera based on the luminescence of luciferin, thereby decoding the DNA sequence.
４．塩基配列データ解析
得られた塩基配列データは、まずＰＨＰで記述したスクリプトにより断片長が２４０塩基以下のもの、平均Ｑｕａｌｉｔｙ ｓｃｏｒｅが２５を下回るものを取り除き、次にＲで記述したスクリプトにより８Ｆプライマーを含まないもの、６塩基を超えるホモポリマーを含むもの、Ｎを含むものを解析から除外した。残りの配列については挿入した６塩基のタグ配列の情報をもとに各被験者に振り分けた。それぞれの配列はＵＣＬＵＳＴを用いて９７％以上の配列相同性を示すものをＯｐｅｒａｔｉｏｎａｌ Ｔａｘｏｎｏｍｉｃ Ｕｎｉｔｓ （ＯＴＵ） としてまとめ、最も高頻度検出された配列を各ＯＴＵの代表配列とした。代表配列はＰｙＮＡＳＴを用いてアラインメントを行った。Ｃｈｉｍｅｒａ Ｓｌａｙｅｒ を用いてキメラ配列を推定し、それらが１検体のみから検出されていた場合キメラ配列として取り除いた。各代表配列はＲＤＰ Ｃｌａｓｓｉｆｉｅｒを用いて属レベルまでその由来を決定した。
検体間の細菌構成類似度はＵｎｉＦｒａｃを用いて評価し、主座標分析を用いて示した。菌叢構成の複雑さについては検出ＯＴＵ数およびＳｈａｎｎｏｎ ｄｉｖｅｒｓｉｔｙ ｉｎｄｅｘをＲを用いて算出し評価した。
4). Base sequence data analysis The base sequence data obtained was first removed from scripts with a fragment length of 240 bases or less, with an average quality score of less than 25 using a script described in PHP, and then with an 8F primer using a script described in R. Those not containing, those containing homopolymers exceeding 6 bases, and those containing N were excluded from the analysis. The remaining sequences were assigned to each subject based on the information of the inserted 6-base tag sequence. For each sequence, those showing a sequence homology of 97% or more using UCLUST were compiled as Operational Taxonomic Units (OTU), and the most frequently detected sequence was used as a representative sequence of each OTU. The representative sequence was aligned using PyNAST. Chimera sequences were estimated using Chimera Layer and removed as chimeric sequences if they were detected from only one specimen. The origin of each representative sequence was determined to the genus level using RDP Classifier.
Bacterial structural similarity between specimens was evaluated using UniFrac and indicated using principal coordinate analysis. The complexity of the bacterial flora composition was evaluated by calculating the number of detected OTUs and the Shannon diversity index using R.
５．結果
菌叢の細菌構成類似度を、ＵｎｉＦｒａｃを用いて解析した結果を図２に示す。図２中、三角は齲蝕経験群の唾液サンプルの結果、丸は齲蝕非経験群由来の唾液サンプルの結果である。齲蝕経験群と、齲蝕非経験群で唾液細菌叢の構成が異なっていることが示された。
菌叢の細菌構成の複雑さを解析した結果を図３に示す。図３Ａは種の豊かさ（Ｓｐｅｃｉｅｓ ｒｉｃｈｎｅｓｓ）で示し，図３Ｂは、多様性指数（Ｓｈａｎｎｏｎ ｄｉｖｅｒｓｉｔｙ ｉｎｄｅｘ）で示す。いずれに指標によっても、齲蝕非経験群では、齲蝕経験群と比較して、細菌構成が複雑であることが示された。この差は、いずれも、Ｔ検定によりＰ＜０．０５と有意であった。
5. Result The result of having analyzed the bacterial composition similarity of the flora using UniFrac is shown in FIG. In FIG. 2, triangles are the results of saliva samples from the caries experience group, and circles are the results of saliva samples from the caries inexperienced group. It was shown that the salivary bacterial flora was different between the caries-experienced group and the caries-inexperienced group.
The result of analyzing the complexity of the bacterial composition of the flora is shown in FIG. FIG. 3A shows the species richness (species richness), and FIG. 3B shows the diversity index (Shannon diversity index). In any case, the bacterial composition was shown to be more complicated in the caries-inexperienced group than in the caries-experienced group. This difference was significant as P <0.05 by T test.
菌属については、全ての試料で検出されたのは７３菌属であった。そのうち１０菌属は、全ての被験者から検出された。また、齲蝕経験の有無に関わらず、Ｓｔｒｅｐｔｃｏｃｃｕｓが最も優勢であった。それぞれの群で優勢な菌属の結果を図４に示す。図４Ａは齲蝕経験者の唾液細菌叢の菌属、図４Ｂは齲蝕非経験者の唾液細菌叢の菌属を示す。また、齲蝕経験者と、齲蝕非経験者の、唾液中の菌叢構成比率で、特徴的に異なる菌属を図５に示す。図５に示される７菌属において、両群で存在比率が有意に異なっていた（Ｔ検定によりＰ＜０．０５）。
両群を、１０００リードあたりのＯＴＵ数（横軸）と、Ｐｏｒｐｈｙｒｏｍｏｎａｓの割合（縦軸）で展開した結果を図６に示す。齲蝕非経験では、９名中８名が検出ＯＴＵ数２７０以上、Ｐｏｒｐｈｙｒｏｍｏｎａｓ割合が３．５％以上であったのに対し、齲蝕経験群では、そのような被験者は認められなかった。
As for the genus, 73 were detected in all samples. Of these, 10 species were detected from all subjects. In addition, Streptococcus was the most dominant regardless of caries experience. The results for the dominant fungi in each group are shown in FIG. FIG. 4A shows the genus of salivary bacterial flora of caries experienced persons, and FIG. 4B shows the genus of salivary bacterial flora of caries non-experienced persons. In addition, FIG. 5 shows fungal genera that are characteristically different in the salivary bacterial flora composition ratio between caries experienced and caries non-experienced. In 7 genera shown in FIG. 5, the abundance ratio was significantly different in both groups (P <0.05 by T test).
FIG. 6 shows the results of developing both groups with the number of OTUs per 1000 leads (horizontal axis) and the ratio of Porphyromonas (vertical axis). In caries non-experience, 8 out of 9 patients had a detected OTU number of 270 or more and a Porphyromonas ratio of 3.5% or more, whereas no such subject was found in the caries experienced group.
以上より、齲蝕非経験者の唾液の細菌叢は、齲蝕経験者の唾液の細菌叢に比べて、多様な細菌種によって構成されており、また、Ｐｏｒｐｈｙｒｏｍｏｎａｓ，Ｆｕｓｏｂａｃｔｅｒｉｕｍ，Ｇｅｍｅｌｌａなどが、より高い比率で存在することが明らかになった。
これらの細菌がプラークバイオフィルムの形成過程において、Ｓｔｒｅｐｔｏｃｏｃｃｕｓをはじめとする酸性生菌の増殖に拮抗的に働くことで齲蝕感受性に関与している可能性が考えられる。
すなわち、これらの結果より、唾液中の菌叢のＯＴＵ数が高ければ、齲蝕になりにくいといえ、また、Ｐｏｒｐｈｙｒｏｍｏｎａｓ，Ｆｕｓｏｂａｃｔｅｒｉｕｍ，Ｇｅｍｅｌｌａなどが、より高い比率で存在すれば、齲蝕になりにくいと推測することができる。
From the above, the bacterial flora of saliva inexperienced caries is composed of various bacterial species compared to the bacterial flora of saliva in caries experienced persons, and Porphyromonas, Fusobacterium, Gemella, etc. are at a higher ratio. It became clear that it existed.
There is a possibility that these bacteria are involved in caries susceptibility by acting antagonistically to the growth of live acidic bacteria such as Streptococcus during the formation process of plaque biofilm.
That is, from these results, it can be said that if the OTU number of the bacterial flora in saliva is high, it will be difficult to become caries, and if Porphyromonas, Fusobacterium, Gemella, etc. are present at a higher ratio, it will be difficult to caries. can do.
Claims (10)
採取した唾液中のＤＮＡを抽出し、
抽出されたＤＮＡ中の１６Ｓ ｒＲＮＡ遺伝子を増幅し、
増幅された遺伝子の１０００リード以上あたりのＯＴＵ数の配列の解析を行うこと
を特徴とする請求項１または２のいずれかに記載の齲蝕のなりにくさの診断を補助するためのデータ収集方法。 Collecting saliva from the subject,
Extract the DNA in the collected saliva,
Amplifying the 16S rRNA gene in the extracted DNA;
3. The data collection method for assisting diagnosis of the difficulty of caries according to claim 1, wherein the sequence of the number of OTUs per 1000 or more reads of the amplified gene is analyzed.
採取した唾液中のＤＮＡを抽出し、
抽出されたＤＮＡ中の１６Ｓ ｒＲＮＡ遺伝子を増幅し、
増幅された遺伝子の１０００リード以上あたりのＯＴＵ数の配列の解析を行うこと
を特徴とする請求項７から８のいずれかに記載の解析方法。 Collecting saliva from the subject,
Extract the DNA in the collected saliva,
Amplifying the 16S rRNA gene in the extracted DNA;
The analysis method according to any one of claims 7 to 8, wherein a sequence of OTU number per 1000 reads or more of the amplified gene is analyzed.
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