JP4359699B2 - Method for detecting carious bacteria and primer set used in the method - Google Patents

Description

S.sobrinusの検出のためのプライマーセット（プライマーセットB）は、下記のプライマーからなり、常法により合成することができる。

本発明の方法において、「検出」の語は、定量的検出及び定性的検出の両方を意味する。
【００１４】
増幅のためのプライマーとの反応は、例えば上記ＤＮＡ試料１〜２μｌ、プライマー（S.mutansの検出のためには配列番号１〜５の配列を有するプライマー、S.sobrinusの検出のためには配列番号６〜１１の配列を有するプライマー、好ましくはＦ３，Ｂ３については０．１〜０．５μＭ、ＦＩＰ，ＢＩＰについては１．０〜２．０μＭ、ＦＬＰ，ＢＬＰについては０．５〜１．０μＭ）、ｄＮＴＰ各１〜２μＭ、鎖置換型ＤＮＡポリメラーゼ５〜３２Ｕを反応緩衝液中に最終容量が２５μｌとなるように混合し、好ましくは６５℃で、好ましくは１０〜４０分間、より好ましくは１５〜３０分間保温することにより行われる。
【００１５】
増幅されたＤＮＡの検出は、例えば、ＤＮＡの増幅の副産物として生じるピロリン酸マグネシウムによる白濁の程度を測定することにより行うことができる。なお、白濁の程度または有無の判定は目視により行うこともできる。また、増幅産物の検出は、電気泳動により行うこともできる。さらに、増幅を蛍光インターカレーターの存在下で行い、蛍光強度を測定することにより行うこともできる。
【００１６】
判定はう触細菌の数が、１０⁶個未満であるか、または１０⁶個以上であるかで行う。１０⁶個以上である場合には口腔内がう蝕になりやすい環境にあると判定される。この場合、患者にう触になりやすいことを認識してもらい、患者口腔内清掃意識の向上をねらったり、う触予防処置を施すことにより、う蝕を予防することができる。また、将来的には検出された細菌を特異的に抑制する予防処置も考えられている。
【００１７】
本発明の方法は、さらに、上記う蝕細菌の検出結果に基づき、被験者の口腔内のう蝕の状態を判定する工程を含むこともできる。
本発明は、上記プライマーセットAまたはBを含むキットに関する。本発明のキットは、さらに鎖置換型ＤＮＡポリメラーゼ、緩衝液、ｄＮＴＰ等を含んでいてもよい。
本発明のキットは、例えば、口腔内のう触細菌による汚染状況の判定、う触リスクの診断、さらに予防処置法の選択の判断基準のためのものである。
【００１８】
【実施例】
以下の実施例は、本発明を説明するためのものであり、本発明はこれに限定されることはない。
実施例１ 爪楊枝にて採取した歯垢中のS.mutans菌の検出
爪楊枝によりヒトの歯垢を採取し、これを２００μｌの溶菌液（１０ｍＭのTris-HCl緩衝液、1mMのＥＤＴＡ、１％のTritonX-100，pH８）に入れ、激しく混和し、１０分間煮沸した後、遠心分離し、その上清を試料とした。
【００１９】
前述のプライマー、SmuF3（配列番号１）、SmuB3（配列番号２）、SmuFIP（配列番号３）、SmuBIP（配列番号４）、SmuLB（配列番号５）は、北海道システムサイエンス社のカスタムオリゴＤＮＡサービスにより合成したものを使用した（プライマーセットA）。
上記で調製した試料溶液２μｌ、プライマーSmuF3０．２μＭ、プライマーSmuB3 ０．２μＭ、プライマーSmuFIP１．６μＭ、プライマーSmuBIP１．６μＭ及びプライマーSmuLP０．８μＭ、BstＤＮＡポリメラーゼ（New England Biolabs）８Ｕnit及び２０ｍＭ TrisHCl(pH8.8)(和光純薬)、１０ｍＭ ＫＣｌ（和光純薬）、８ｍＭのＭｇＳＯ₄（和光純薬）、１０ｍM（NH₄）₂SO₄（和光純薬）0.1%Tween 20（和光純薬）含有するThermopol緩衝液）、０．８Ｍのベタイン（Aldrich Chemical）、２ｍMのMgSO₄（Nakalai Tesque）、各１．４ｍＭのｄＡＴＰ、ｄＴＴＰ、ｄＧＴＰ及びｄＣＴＰを透明マイクロチューブ（ABgene製）に入れて混合し、６５℃で３０分間インキュベートすることにより、S.mutansのgtfB遺伝子内の標的配列（配列表の配列番号１２の２２５５〜２３２５の配列）を増幅した。
【００２０】
増幅後のマイクロチューブ内の溶液を目視したところ、白濁しており、ＤＮＡが増幅された。即ち、試料中にS.mutansが存在することが確認された。
試験例 １
本発明の検出方法の感度を調べるために、各濃度のS.mutansの試料溶液及びS.sobrinusの試料溶液を調製し、本発明の方法及び従来のＰＣＲ法により検出した。
【００２１】
（１）S.mutansのＤＮＡ溶液の調製
S. mutans Xc株（九州大学大学院歯学研究院口腔保健推進学講座より供与）の染色体ＤＮＡは、Jagusztyn-Krynickaら（1982）によって報告されている方法に基づいて調製した。S. mutansは、THブロスを用いて37℃で培養し、対数増殖期（550 nmでの吸光度が約0.5）にある菌液10 mlに20%グリシンを2.5 ml加えてさらに37℃で60分間培養した。菌体を遠沈によって回収し、TE緩衝液[1 mMエチレンジアミン四酢酸（EDTA）を含む10 mMトリス塩酸塩、pH 8.0]で遠沈洗浄後、菌体を20%グルコースを含むTE緩衝液250μlに浮遊させた。その菌体浮遊液に1 mg/mlのムタノリシンK-1（Yokogawaら、1974；Hamadaら、1978）を25 μl加えて65℃で20分間反応させた。ついで、10 mg/mlのリゾチーム（和光純薬、大阪）を100μl加えて42℃で40分間反応させた後、0.5 M EDTAを100μlと10 mg/mlのリボヌクレアーゼ（和光純薬）を2μl加えて37℃で30分間保温した。さらに、20 mg/mlのプロテイナーゼK（Sigma, St. Louis, Mo., USA）を5μl加えて37℃で60分間反応させた後、10%SDS溶液を100μl加えて37℃で30分間保温した。こうしてS. mutansを溶菌させた液に、剪断力がかからないように穏やかにフェノール・クロロホルム処理を水層がほぼ透明になるまで行い、最終的に単離した水層に等容量のイソプロパノールと1/10容量の3 M酢酸ナトリウム溶液（pH 5.2）を加えて穏やかに混和した。この処理で不溶化したＤＮＡをガラス棒で巻き取り、1.0 mlの70%冷エタノールで洗浄した後、室温で自然乾燥し、最後に100 μlのTE緩衝液に溶解して染色体ＤＮＡを得た。
【００２２】
（２）S.sobrinusのＤＮＡ溶液の調製
S. mutansの代わりに、S. sobrinus OMZ176株（九州大学大学院歯学研究院口腔保健推進学講座より供与）を用いること以外は、上記S. mutansのＤＮＡ溶液の調製方法と同じ方法にてS.sobrinusのＤＮＡ 溶液を調製した。
【００２３】
（３）本発明の方法による増幅
試料として、爪楊枝抽出物の代わりに上記で調製したS.mutansのＤＮＡ溶液またはS.sobrinusのＤＮＡ溶液を用いること以外は、実施例１と同じ方法により、増幅を行った。なお、S.mutansの検出のためには、前述のプライマーセットAを実施例１に示した量で用い、S.sobrinusの検出のためには、前述のプライマー、SsobF3（配列番号６）、SsobB3（配列番号７）、SsobFIP（配列番号８）、SsobBIP（配列番号９）、SsobLF（配列番号１０）、SsobLB（配列番号１１）からなるプライマーセットBを試料溶液２μｌに対してプライマーSsobF3 ０．２μＭ、プライマーSsobB3 ０．２μＭ、プライマーSsobFIP １．６μＭ、プライマーSsobBIP １．６μＭ及びプライマーSsobLF ０．８μＭ 及びSsobBF ０．８μMの量で用いた。
【００２４】
（４）ＰＣＲによる増幅
１０ｍＭTris-HCl緩衝液（ｐH8.3）、１．５ｍMのMgCl₂、５０ｍMのKCl、各２００μMのｄATP、ｄTTP、ｄGTP及びｄCTP、１μMの下記のプライマー（GTFB-F，-R）、１UのExTaqＤＮＡポリメラーゼ（宝酒造（株））及び２μｌの上記のS.mutansのＤＮＡ溶液またはS.sobrinusのＤＮＡ溶液からなるＰＣＲ反応液を用い、９５℃で３０秒間５９℃で３０秒間、７２℃で１分間を３０サイクルのＰＣＲをでＰＣＲを行うことにより、S.mutansのgtfB遺伝子の一部を増幅した。
プライマーとしてGTFB-F，GTFB-Rの代わりに下記のGTFI-F，GTFI-Rを用いてS.sobrinusのgtfI遺伝子の一部を増幅した。

【００２５】
（５）検出
増幅産物を２％アガロース電気泳動にかけ（約２０分間）、比較した。結果を図2（S.mutans）及び図３（S.sobrinus）に示す。
図より、S.mutansのＤＮＡを本発明の方法で増幅した場合、ＰＣＲで増幅した場合に比べて検出感度が約１０倍となることがわかる。
また、S.sobrinusのＤＮＡを本発明の方法で増幅した場合、ＰＣＲで増幅した場合に比べて検出感度が約１００倍となることがわかる。
【００２６】
試験例２
試料として、S.mutans染色体ＤＮＡを１ｍｌ中に１０³，１０⁵，１０⁶個相当分含む溶液を用い、増幅のための反応時間を１０分間、１５分間、２０分間、２５分間、３０分間または３５分間とした以外は、実施例１と同様の方法により、S.mutansのgtfB遺伝子を増幅し、白濁の有無を目視により判定した。結果を下記の表に示す。表中、＋は白濁が観察されたことを示し、−は白濁が観察されなかったことを示す。
【００２７】

【００２８】
上表より、試料中のS.mutansのＤＮＡ濃度と白濁が観察される反応時間との間に相関関係があることがわかる。従って、試料溶液中のS.mutansのおおよその量を白濁が生じる反応時間により知ることができる。また、唾液１ｍｌ中、S.mutansが１０⁶以上である場合、う触発生の可能性が高いため、この試験例の測定条件においては、反応時間を１５分とすることにより、増幅産物の白濁の有無を目視することにより、高度にう触の生じやすい状態であるか否かを判定することができる。
【００２９】
試験例３
試料として、爪楊枝により採取した歯垢試料の代わりに、S.mutans Xc、S.mutans MT8148、S.mutans LM7、S.mutans MT6219、S.sobrinus OMZ176、S.sobrinus PMZ65、S.sobrinus MT8245、S.salivarius HHT、S.salivarius HT9R、S.sangius ATCC10556、S.gordonii Challis、E.coli DH5a、A. actinomycetemcomitans Y4及びP.glugivalis W83（上記菌株は全て九州大学大学院歯学研究院口腔保険推進学講座より供与された）を用い、上記実施例１の方法により、上記プライマーセットAを用いた増幅（結果を図４に示す）及び上記プライマーセットBを用いた増幅（結果を図５に示す）を行った。
図より明らかなように、プライマーセットAを用いるとS.mutansのＤＮＡが特異的に増幅され、プライマーセットＢを用いるとS.sobrinusのＤＮＡが特異的に増幅される。
【００３０】
【発明の効果】
本発明により、う触細菌であるS.mutans及びS.sobrinusの各々が簡便且つ迅速に、しかも高い精度で検出することができる。しかも増幅反応を一定温度で行うことができるため、高価な機器を必要とすることもない。これにより、臨床においてこれらのう触細菌による口腔内の汚染状況を検査することが可能となり、歯科治療において予防及び治療に役立てることが可能になる。また、ＰＣＲにて増幅の難しい爪楊枝で採取した試料においても本発明による方法では増幅が可能であり、公衆におけるう触細菌の調査や予防意識の向上にも役立てることができる。
【００３１】
【配列表】

【図面の簡単な説明】
【図１】 本発明の方法に用いるＬＡＭＰ法を説明する模式図である。
【図２】 本発明の方法及びＰＣＲ法による増幅産物の電気泳動分析の結果を示す写真である。
【図３】 本発明の方法及びＰＣＲ法による増幅産物の電気泳動分析の結果を示す写真である。
【図４】 本発明の方法による増幅産物の電気泳動分析の結果を示す写真である。
【図５】 本発明の方法による増幅産物の電気泳動分析の結果を示す写真である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for detecting carious bacteria, a primer set used in the method, and a kit for detecting carious bacteria containing the primer set.
[0002]
[Prior art]
Streptococcus mutans (S.mutans) and Streptococcus sobrinus (S.sobrinus) are known as bacteria that cause touch. By detecting these bacteria in the patient's oral cavity in the early stages of caries and determining the risk of dental caries, it is considered to be useful for planning caries prevention plans and instructing patients. . It is also considered to use it as information for caries treatment.
At present, these bacteria are mainly detected by a culture method.
Detection by the PCR method has also been developed.
[0003]
[Problems to be solved by the invention]
However, the culture method has a drawback that it takes several days to culture.
In addition, the PCR method requires expensive equipment and takes several hours to make a determination, so it is not practical as a method for use in a clinical field. In addition, it is desirable to collect samples in the oral cavity with toothpicks that are inexpensive and do not require operations such as sterilization when performing similar touching risk determinations in the public or conducting epidemiological surveys, etc. The PCR reaction is not successful with the sample collected in this way.
[0004]
The present inventors have recently developed a loop-mediated isothermal amplification method (Tsugunori Notomi et al. Loop-mediated Isothermal Amplification of DNA, Nucleic Acids Research 28, e63 (2000); Yasuyoshi Mori et al., Detection of Loop- mediated Isothermal Amplification Reaction by Turbidity Derived from Magnesium Pyrophosphate Formation, Biochemical and Biophysical Research Communications 289, 150-154 (2001); K. Nagamine et al., Accelerated reaction by loop-mediated isothermal amplification using loop primers, Molecular and Cellular Probes (2002 ) 16, 223-229), and a method for detecting each of S. mutans and S. sobrinus easily and quickly was found by amplifying specific sites of DNA of S. mutans and S. sobrinus.
[0005]
[Means for Solving the Problems]
(1) The present invention relates to a step of preparing a DNA sample from a sample collected from the oral cavity of a subject, and the DNA sample in the presence of a strand displacement type DNA synthase. And detection of Streptococcus.mutans in the oral cavity, comprising a step of amplifying the target DNA by reacting with a primer set comprising a primer having the sequence of 5 and a constant time at a constant temperature, and a step of detecting the obtained amplification product Regarding the method.
[0006]
(2) The present invention also includes a step of preparing a DNA sample from a sample collected from the oral cavity of a subject, and the DNA sample is prepared in the presence of a strand displacement type DNA synthase, respectively in SEQ ID NOs: 6, 7, and 8 in the sequence listing. , Including a step of amplifying a target DNA by reacting with a primer set comprising a primer having the sequence of 9, 10, and 11 at a constant reaction temperature for a predetermined time, and a step of detecting the obtained amplification product About the detection method of Streptococcus.sobrinus.
[0007]
(3) Further, in the method of (1) or (2) above, the present invention comprises the step of preparing the DNA sample, wherein a sample collected from the oral cavity is treated with a lysate and then heated at 80 ° C. to boiling temperature. It is related with the method characterized by performing by centrifuging after collecting for 30 minutes after heating, and extract | collecting the supernatant liquid.
(4) Furthermore, the present invention relates to the method of (1) to (3) above, wherein the reaction temperature is a single temperature of 65 ° C. in the step of amplifying the target DNA.
[0008]
(5) Furthermore, the present invention relates to a primer set for detecting Streptococcus.mutans, which comprises primers each having a sequence set forth in SEQ ID NOs: 1 to 5 in the sequence listing.
(6) Furthermore, the present invention relates to a primer set for detecting Streptococcus.sobrinus comprising primers each having the sequences described in SEQ ID NOs: 6 to 11 in the sequence listing.
(7) Furthermore, the present invention relates to a kit for detecting Streptococcus.mutans comprising the primer set of (5).
(8) Further, the present invention relates to a kit for detecting Streptococcus.sobrinus comprising the primer set of (6).
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The method of the present invention relates to a method for detecting S. mutans and S. sobrinus using a Loop-mediated Isothermal Amplification method (LAMP method).
As shown in FIG. 1, the LAMP method defines three regions F3c, F2c, and F1c from the 3 ′ end side to the target DNA sequence, and three regions B1, B2, and B3 from the 5 ′ end side. A method for designating four types of primers, FIP, F3, BIP and B3, and further designing LoopPrimerB and / or LoopPrimerF as desired, performing amplification of these DNAs, and detecting amplification products It is. The FIP primer has an F2 region complementary to the F2c region on the 3 ′ end side and the same sequence as the F1c region on the 5 ′ end side. The F3 primer has an F3 region complementary to the F3c region of the target gene. The BIP primer has a region complementary to the B2c region of the target gene on the 3 ′ end side and the same sequence as B1c on the 5 ′ end side. The B3 primer has a sequence complementary to the B3c region. LoopPrimerB has a sequence complementary to the sequence between the B1 region and B2 region, and LoopPrimerF has a sequence complementary to the sequence between the F1 region and F2 region.
[0010]
When these primers are reacted at a temperature of 60 to 65 ° C. in the presence of a strand displacement type DNA synthase and dNTP, an amplification product having a structure in which complementary sequences repeat on the same strand can be obtained. This method is superior to the PCR method in that the reaction may be performed at a constant temperature and amplification is possible in a short time.
In the method of the present invention, the sample collected from the subject's mouth is, for example, saliva, plaque, and the like.
[0011]
Saliva may be collected after chewing paraffin or the like.
For example, plaque is collected using a probe or a toothpick.
For preparation of the DNA sample, for example, 10 to 100 μl of lysate is added to a sample collected from the oral cavity of the subject and mixed, and then 80 ° C. to boiling temperature, preferably 1 to 30 minutes at boiling temperature, preferably 5 to 5 minutes. This can be done by heating for 15 minutes and collecting the centrifuged supernatant.
[0012]
In the method of the present invention, in the case of detecting S. mutans, a part of the sequence (base) of the target DNA, that is, the glucosyltransferase gene of S. mutans (gtfB, GenBank accession number M1736, described in SEQ ID NO: 12 in the sequence listing). No. 2255-2325) is amplified. In the case of detecting S. sobrinus, a part of the sequence of the target DNA, that is, the glucosyltransferase gene of S. sobrinus (gtfI, GenBank accession number D90213, described in SEQ ID NO: 13 in the sequence listing) (base numbers 2311 to 2382) is used. Amplify.
[0013]
A primer set (primer set A) for detection of S. mutans comprises the following primers and can be synthesized by a conventional method.

The primer set (primer set B) for detecting S. sobrinus comprises the following primers and can be synthesized by a conventional method.

In the method of the present invention, the term “detection” means both quantitative detection and qualitative detection.
[0014]
The reaction with the primer for amplification includes, for example, 1 to 2 μl of the above DNA sample, a primer (a primer having the sequence of SEQ ID NO: 1 to 5 for detection of S. mutans, a sequence for detection of S. sobrinus Primers having the sequences of numbers 6 to 11, preferably 0.1 to 0.5 μM for F3 and B3, 1.0 to 2.0 μM for FIP and BIP, 0.5 to 1.0 μM for FLP and BLP ), 1 to 2 μM each of dNTP and 5 to 32 U of strand displacement DNA polymerase are mixed in the reaction buffer so that the final volume is 25 μl, preferably at 65 ° C., preferably 10 to 40 minutes, more preferably 15 It is performed by keeping warm for -30 minutes.
[0015]
The amplified DNA can be detected, for example, by measuring the degree of white turbidity caused by magnesium pyrophosphate generated as a by-product of DNA amplification. The degree of white turbidity or the presence / absence can also be determined visually. The amplification product can also be detected by electrophoresis. Furthermore, amplification can be performed in the presence of a fluorescence intercalator and the fluorescence intensity can be measured.
[0016]
Determining the number of caries bacteria, carried out in either a 10 less than ^six or where 10 ⁶ or more. When the number is 10 ⁶ or more, it is determined that the oral cavity is in an environment that tends to cause caries. In this case, caries can be prevented by having the patient recognize that it is likely to touch and aiming to improve the patient's oral cleaning awareness or by performing a touch prevention treatment. In the future, prophylactic treatment for specifically suppressing the detected bacteria is also considered.
[0017]
The method of the present invention may further include a step of determining the state of caries in the oral cavity of the subject based on the detection result of the carious bacteria.
The present invention relates to a kit containing the primer set A or B. The kit of the present invention may further contain a strand displacement type DNA polymerase, a buffer, dNTP and the like.
The kit of the present invention is used, for example, for determination of contamination status by oral bacteria in the oral cavity, diagnosis of contact risk, and determination criteria for selecting a preventive treatment method.
[0018]
【Example】
The following examples are for explaining the present invention, and the present invention is not limited thereto.
Example 1 Detection of S. mutans bacteria in dental plaque collected with toothpick Human dental plaque was collected with toothpick, and 200 μl of lysate (10 mM Tris-HCl buffer, 1 mM EDTA, 1% TritonX-100, pH 8), vigorously mixed, boiled for 10 minutes, centrifuged, and the supernatant was used as a sample.
[0019]
The above-mentioned primers, SmuF3 (SEQ ID NO: 1), SmuB3 (SEQ ID NO: 2), SmuFIP (SEQ ID NO: 3), SmuBIP (SEQ ID NO: 4), and SmuLB (SEQ ID NO: 5) are available from the custom oligo DNA service of Hokkaido System Science. The synthesized one was used (Primer Set A).
2 μl of the sample solution prepared above, Primer SmuF3 0.2 μM, Primer SmuB3 0.2 μM, Primer SmuFIP 1.6 μM, Primer SmuBIP 1.6 μM and Primer SmuLP 0.8 μM, Bst DNA polymerase (New England Biolabs) 8 Unit and 20 mM TrisHCl (pH 8.8) (Wako Pure Chemical) Thermopol buffer containing 10 mM KCl (Wako Pure Chemical), 8 mM MgSO ₄ (Wako Pure Chemical), 10 mM (NH ₄ ) ₂ SO ₄ (Wako Pure Chemical) 0.1% Tween 20 (Wako Pure Chemical) Solution), 0.8 M betaine (Aldrich Chemical), 2 mM MgSO ₄ (Nakalai Tesque), 1.4 mM each of dATP, dTTP, dGTP and dCTP in a clear microtube (ABgene) and mixed at 65 ° C. Was amplified for 30 minutes to amplify the target sequence (sequence 2255 to 2325 of SEQ ID NO: 12 in the sequence listing) in the gtfB gene of S. mutans.
[0020]
When the solution in the microtube after amplification was visually observed, it was cloudy and DNA was amplified. That is, it was confirmed that S.mutans was present in the sample.
Test example 1
In order to examine the sensitivity of the detection method of the present invention, sample solutions of S. mutans and S. sobrinus at various concentrations were prepared and detected by the method of the present invention and the conventional PCR method.
[0021]
(1) Preparation of DNA solution of S.mutans
Chromosomal DNA of S. mutans Xc strain (provided by the Department of Oral Health Promotion, Kyushu University Graduate School of Dentistry) was prepared based on the method reported by Jagusztyn-Krynicka et al. (1982). S. mutans is cultured in TH broth at 37 ° C, 2.5 ml of 20% glycine is added to 10 ml of the bacterial solution in the logarithmic growth phase (absorbance at 550 nm is about 0.5), and further at 37 ° C for 60 minutes. Cultured. The cells are collected by centrifugation, and after centrifugation with TE buffer [10 mM Tris hydrochloride containing 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0], the cells are washed with 250 μl of TE buffer containing 20% glucose. Floated on. 25 μl of 1 mg / ml mutanolysin K-1 (Yokogawa et al., 1974; Hamada et al., 1978) was added to the cell suspension and reacted at 65 ° C. for 20 minutes. Next, after adding 100 μl of 10 mg / ml lysozyme (Wako Pure Chemicals, Osaka) and reacting at 42 ° C. for 40 minutes, 100 μl of 0.5 M EDTA and 2 μl of 10 mg / ml ribonuclease (Wako Pure Chemicals) were added. Incubated at 37 ° C for 30 minutes. Further, 5 μl of 20 mg / ml proteinase K (Sigma, St. Louis, Mo., USA) was added and reacted at 37 ° C. for 60 minutes, then 100 μl of 10% SDS solution was added and incubated at 37 ° C. for 30 minutes. . The solution obtained by lysing S. mutans was gently treated with phenol / chloroform so that no shear force was applied until the aqueous layer became almost transparent, and finally the isolated aqueous layer was mixed with an equal volume of isopropanol and 1 / 10 volumes of 3 M sodium acetate solution (pH 5.2) was added and gently mixed. The DNA insolubilized by this treatment was wound up with a glass rod, washed with 1.0 ml of 70% cold ethanol, air dried at room temperature, and finally dissolved in 100 μl of TE buffer to obtain chromosomal DNA.
[0022]
(2) Preparation of DNA solution of S. sobrinus
Instead of S. mutans, S. sobrinus OMZ176 strain (provided by Kyushu University Graduate School of Dentistry, Department of Oral Health Promotion) was used, and S. mutans DNA solution was prepared in the same manner as described above. A DNA solution of sobrinus was prepared.
[0023]
(3) Amplification by the same method as in Example 1 except that the S.mutans DNA solution or S.sobrinus DNA solution prepared above was used instead of the toothpick extract as the amplification sample by the method of the present invention. Went. For detection of S. mutans, the aforementioned primer set A was used in the amount shown in Example 1, and for detection of S. sobrinus, the aforementioned primers, SsobF3 (SEQ ID NO: 6), SsobB3 Primer set B consisting of (SEQ ID NO: 7), SsobFIP (SEQ ID NO: 8), SsobBIP (SEQ ID NO: 9), SsobLF (SEQ ID NO: 10), SsobLB (SEQ ID NO: 11) was added to 2 μl of the sample solution as primer SsobF3 0.2 μM. , Primer SsobB3 0.2 μM, primer SsobFIP 1.6 μM, primer SsobBIP 1.6 μM and primers SsobLF 0.8 μM and SsobBF 0.8 μM.
[0024]
(4) Amplification by PCR 10 mM Tris-HCl buffer (pH 8.3), 1.5 mM MgCl ₂ , 50 mM KCl, 200 μM each of dATP, dTTP, dGTP and dCTP, 1 μM of the following primers (GTFB-F, − R) A PCR reaction solution consisting of 1 U ExTaq DNA polymerase (Takara Shuzo Co., Ltd.) and 2 μl of the above S.mutans DNA solution or S.sobrinus DNA solution at 95 ° C. for 30 seconds at 59 ° C. for 30 seconds, A part of the gtfB gene of S. mutans was amplified by performing PCR with 30 cycles of PCR for 1 minute at 72 ° C.
A part of the gtfI gene of S. sobrinus was amplified using the following GTFI-F and GTFI-R instead of GTFB-F and GTFB-R as primers.

[0025]
(5) The detection amplification product was subjected to 2% agarose electrophoresis (about 20 minutes) and compared. The results are shown in FIG. 2 (S.mutans) and FIG. 3 (S.sobrinus).
From the figure, it can be seen that when S. mutans DNA is amplified by the method of the present invention, the detection sensitivity is about 10 times higher than when amplified by PCR.
It can also be seen that when S. sobrinus DNA is amplified by the method of the present invention, the detection sensitivity is about 100 times higher than when amplified by PCR.
[0026]
Test example 2
As a sample, a solution containing 10 ³ , 10 ⁵ , 10 ⁶ equivalents of S.mutans chromosomal DNA in 1 ml was used, and the reaction time for amplification was 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes or Except for 35 minutes, the gtfB gene of S. mutans was amplified in the same manner as in Example 1, and the presence or absence of cloudiness was visually determined. The results are shown in the table below. In the table, + indicates that white turbidity was observed, and-indicates that white turbidity was not observed.
[0027]

[0028]
From the above table, it can be seen that there is a correlation between the DNA concentration of S. mutans in the sample and the reaction time at which white turbidity is observed. Therefore, the approximate amount of S. mutans in the sample solution can be known from the reaction time at which white turbidity occurs. In addition, if S.mutans is 10 ⁶ or more in 1 ml of saliva, the possibility of touching is high. Therefore, under the measurement conditions of this test example, the reaction time is set to 15 minutes, so that the amplification product becomes cloudy. By visually observing the presence or absence, it is possible to determine whether or not it is in a state in which touch is highly likely to occur.
[0029]
Test example 3
As a sample, instead of plaque samples collected by toothpick, S.mutans Xc, S.mutans MT8148, S.mutans LM7, S.mutans MT6219, S.sobrinus OMZ176, S.sobrinus PMZ65, S.sobrinus MT8245, S .salivarius HHT, S.salivarius HT9R, S.sangius ATCC10556, S.gordonii Challis, E.coli DH5a, A. actinomycetemcomitans Y4 and P.glugivalis W83 Amplification using the primer set A (results are shown in FIG. 4) and amplification using the primer set B (results are shown in FIG. 5) by the method of Example 1 above. It was.
As is apparent from the figure, when primer set A is used, S. mutans DNA is specifically amplified, and when primer set B is used, S. sobrinus DNA is specifically amplified.
[0030]
【The invention's effect】
According to the present invention, each of the claustrobacterium S.mutans and S.sobrinus can be detected simply, quickly and with high accuracy. Moreover, since the amplification reaction can be performed at a constant temperature, expensive equipment is not required. Thereby, it becomes possible to examine the contamination state in the oral cavity by these touching bacteria in the clinic, and it can be used for prevention and treatment in dental treatment. In addition, a sample collected from a toothpick, which is difficult to amplify by PCR, can be amplified by the method of the present invention, which can be used for investigation of palatine bacteria and improvement of prevention awareness in the public.
[0031]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic diagram illustrating a LAMP method used in the method of the present invention.
FIG. 2 is a photograph showing the results of electrophoretic analysis of an amplification product by the method of the present invention and the PCR method.
FIG. 3 is a photograph showing the results of electrophoretic analysis of an amplification product by the method of the present invention and the PCR method.
FIG. 4 is a photograph showing the result of electrophoretic analysis of an amplification product by the method of the present invention.
FIG. 5 is a photograph showing the result of electrophoretic analysis of an amplification product by the method of the present invention.

Claims (8)

A step of preparing a DNA sample from a sample collected from the oral cavity of the subject, the DNA sample having the sequences described in SEQ ID NOs: 1, 2, 3, 4 and 5 in the sequence listing in the presence of a strand displacement type DNA synthase; A method for detecting Streptococcus.mutans in the oral cavity, comprising a step of amplifying a target DNA by reacting with a primer set consisting of primers at a constant temperature for a predetermined time, and a step of detecting the obtained amplification product. A step of preparing a DNA sample from a sample collected from the oral cavity of the subject, and the DNA sample in the presence of a strand displacement type DNA synthase, each of the sequences described in SEQ ID NOs: 6, 7, 8, 9, 10 and 11 in the sequence listing A method for detecting Streptococcus.sobrinus in the oral cavity comprising a step of amplifying a target DNA by reacting with a primer set comprising a primer having a constant time at a constant reaction temperature, and a step of detecting the obtained amplification product. The step of preparing the DNA sample is performed by treating a sample collected from the oral cavity with a lysate, heating at 80 ° C. to boiling temperature for 1 to 30 minutes, and then centrifuging and collecting the supernatant. The method according to claim 1 or 2, wherein: The method according to any one of claims 1 to 3, wherein a reaction temperature is a single temperature of 65 ° C in the step of amplifying the target DNA. A primer set for detecting Streptococcus.mutans, each comprising a primer having the sequence set forth in SEQ ID NOs: 1 to 5 in the sequence listing. A primer set for detection of Streptococcus.sobrinus, comprising primers each having a sequence set forth in SEQ ID NOs: 6 to 11 in the Sequence Listing. A kit for detecting Streptococcus.mutans comprising the primer set of claim 5. A kit for the detection of Streptococcus.sobrinus comprising the primer set of claim 6.

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