JP5047450B2 - CYP2C19 * 3 allele detection method and nucleic acid probe therefor - Google Patents
CYP2C19 * 3 allele detection method and nucleic acid probe therefor Download PDFInfo
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Description
本発明は、シトクロムP450の分子種CYP2C19の遺伝子の変異の検出法およびそのための核酸プローブに関する。 The present invention relates to a method for detecting a mutation in a gene of molecular species CYP2C19 of cytochrome P450 and a nucleic acid probe therefor.
CYP2C19は、さまざまな薬剤を代謝する酵素であり、たとえば、抗てんかん薬であるメフェニトイン、消化性潰瘍用剤であるオメプラゾール、マラリア薬であるプログアニル、抗うつ剤であるクロミプラミン、等の薬物を代謝する。 CYP2C19 is an enzyme that metabolizes various drugs.For example, mephenytoin, an antiepileptic drug, omeprazole, a peptic ulcer agent, proguanil, a malaria drug, clomipramine, an antidepressant, etc. Metabolize.
CYP2C19の遺伝子のcDNAの636位のGがAに置換する変異（G636A変異）が存在すると、翻訳の際に終止コドンに変化しタンパク質の構造が異常になり、したがって、G636A変異がホモで存在すると、様々な薬剤の代謝効率が減少し副作用等のリスクが上昇する(非特許文献１)。G636A変異を有する遺伝子はCYP2C19*3アレルと呼ばれている。 If there is a mutation that replaces G at position 636 in the cDNA of the CYP2C19 gene with A (G636A mutation), the translation changes into a stop codon during translation, and the structure of the protein becomes abnormal. Therefore, if the G636A mutation exists in homology The metabolic efficiency of various drugs decreases, and the risk of side effects and the like increases (Non-patent Document 1). A gene having a G636A mutation is called a CYP2C19 * 3 allele.
G636A変異が存在するとその部分の制限酵素の認識部位が消失するため、PCRで変異部分を含むように増幅を行い、制限酵素で切断し、その後電気泳動で切断されたかどうかを検出するという方法（PCR-RFLP）で検出を行うことが出来る。 When the G636A mutation is present, the restriction enzyme recognition site disappears, so amplification is performed to include the mutation part by PCR, the restriction enzyme is cleaved, and then it is detected whether it has been cleaved by electrophoresis ( PCR-RFLP) can be used for detection.
PCRは数分子の鋳型から数10億倍もの分子を増幅するため、増幅産物がほんの少し混入した場合でも偽陽性、偽陰性の原因になり得る。PCR-RFLPはPCR反応後に増幅産物を取り出して制限酵素処理を行うという必要があるため、増幅産物が次の反応系に混入する恐れがある。よって、偽陽性、偽陰性の結果が得られてしまうことがある。さらに、PCR終了後、制限酵素で処理を行い、その後電気泳動を行うため、検出に必要な時間も非常に長くかかってしまう。また、操作が複雑なため、自動化が困難である。 Since PCR amplifies billions of times from several molecules of template, even a small amount of amplified product can cause false positives and false negatives. Since PCR-RFLP needs to take out the amplification product after PCR reaction and perform restriction enzyme treatment, the amplification product may be mixed into the next reaction system. Therefore, false positive and false negative results may be obtained. In addition, after PCR is completed, treatment with a restriction enzyme is performed, and then electrophoresis is performed. Therefore, it takes a very long time for detection. Moreover, since the operation is complicated, automation is difficult.
一方、一般に、変異を含む領域をPCRで増幅した後、蛍光色素で標識された核酸プローブを用いて融解曲線分析を行い、融解曲線分析の結果に基づいて変異を解析する方法が知られている（非特許文献２、特許文献１）。 On the other hand, a method is generally known in which a region containing a mutation is amplified by PCR, a melting curve analysis is performed using a nucleic acid probe labeled with a fluorescent dye, and the mutation is analyzed based on the result of the melting curve analysis. (Non-patent document 2, Patent document 1).
本発明の課題は、G636A変異を検出するのに有効な消光プローブを特定し、G636A変異を検出する方法を提供することを課題とする。 An object of the present invention is to identify a quenching probe effective for detecting the G636A mutation and to provide a method for detecting the G636A mutation.
上述のプローブを用いる方法に関する文献においては、プローブの設計に関し、末端部が蛍光色素により標識された消光プローブが標的核酸にハイブリダイゼーションしたとき、末端部分においてプローブ−核酸ハイブリッドの複数塩基対が少なくとも一つのGとCの
ペアを形成するように設計するという教示があるのみである。本発明者らは、G636A変異に関し、上記条件を満たす消光プローブを設計し、検出を試みたが、容易に検出を可能とする消光プローブは得られなかった。
In the literature on the method using a probe, regarding the probe design, when a quenching probe labeled with a fluorescent dye at the end is hybridized to a target nucleic acid, at least one probe-nucleic acid hybrid has at least one base pair at the end. There is only teaching to design to form two G and C pairs. The inventors of the present invention designed a quenching probe that satisfies the above conditions for the G636A mutation and attempted detection, but no quenching probe that enables easy detection was obtained.
本発明者らは、G636A変異を含む特定の領域に基づいて消光プローブを設計することにより、消光プローブを用いる融解曲線分析によりG636A変異を検出できることを見出し、本発明を完成した。
本発明は、以下のものを提供する。
The present inventors have found that the G636A mutation can be detected by melting curve analysis using a quenching probe by designing a quenching probe based on a specific region containing the G636A mutation.
The present invention provides the following.
（１）末端が蛍光色素で標識され、ハイブリダイゼーションしたときに蛍光色素の蛍光が減少する核酸プローブであって、配列番号１に示す塩基配列において塩基番号306で終わる10〜30塩基長の塩基配列を有し、３’末端が蛍光色素で標識されている、または、配列番号２に示す塩基配列において塩基番号296から始まる10〜30塩基長の塩基配列を有し、５’末端が蛍光色素で標識されている前記核酸プローブ。 (1) A nucleic acid probe whose end is labeled with a fluorescent dye and whose fluorescence decreases when hybridized, and has a base sequence of 10 to 30 bases ending with base number 306 in the base sequence shown in SEQ ID NO: 1. 3 'end is labeled with a fluorescent dye, or the base sequence shown in SEQ ID NO: 2 has a base sequence of 10 to 30 bases starting from base number 296, and the 5' end is fluorescent dye The nucleic acid probe being labeled.
（２）核酸プローブが、配列番号８または９に示す塩基配列を有する（１）の核酸プローブ。 (2) The nucleic acid probe according to (1), wherein the nucleic acid probe has the base sequence shown in SEQ ID NO: 8 or 9.
（３）一塩基多型の部位を有する核酸について、蛍光色素で標識された核酸プローブを用いて、蛍光色素の蛍光を測定することにより融解曲線分析を行い、融解曲線分析の結果に基づいて変異を検出する方法であって、一塩基多型は、CYP2C19遺伝子のcDNAにおける636位の変異であり、核酸プローブは、（１）または（２）の核酸プローブである前記方法。 (3) Using a nucleic acid probe labeled with a fluorescent dye, the nucleic acid having a single nucleotide polymorphism site is subjected to melting curve analysis by measuring the fluorescence of the fluorescent dye, and the mutation is based on the result of the melting curve analysis. Wherein the single nucleotide polymorphism is a mutation at position 636 in the cDNA of the CYP2C19 gene, and the nucleic acid probe is the nucleic acid probe of (1) or (2).
本発明によれば、G636A変異を検出するのに有効な消光プローブが提供され、さらに、それを用いるG636A変異を検出する方法およびそのためのキットが提供される。G636A変異はCYP2C19*3アレルはG636A変異を含むので、本発明の消光プローブならびに方法およびキットは、CYP2C19*3アレルの検出に使用できる。 According to the present invention, a quenching probe effective for detecting the G636A mutation is provided, and further, a method for detecting the G636A mutation using the same and a kit for the same are provided. Since the G636A mutation contains the G636A mutation in the CYP2C19 * 3 allele, the quenching probes and methods and kits of the present invention can be used to detect the CYP2C19 * 3 allele.
Tm解析は数十秒で完了するため、検出に必要な時間が大幅に短縮出来る。プローブの存在下での核酸の増幅とTm解析を組み合わせる本発明の好ましい態様によれば、核酸の増幅後にプローブのTmを解析するだけなので、反応終了後増幅産物を取り扱う必要がない。よって、増幅産物による汚染の心配がない。また、さらに、増幅に必要な機器と同じ機器で検出することが可能なので、容器を移動する必要すらない。よって、自動化も容易である。 Since Tm analysis is completed in tens of seconds, the time required for detection can be greatly reduced. According to a preferred embodiment of the present invention that combines nucleic acid amplification and Tm analysis in the presence of a probe, it is only necessary to analyze the Tm of the probe after amplification of the nucleic acid, so there is no need to handle the amplification product after the reaction is complete. Therefore, there is no worry of contamination by amplification products. Furthermore, since it can be detected by the same equipment as that required for amplification, it is not necessary to move the container. Therefore, automation is also easy.
本発明プローブは、末端が蛍光色素で標識され、ハイブリダイゼーションしたときに蛍光色素の蛍光が減少する核酸プローブであって、配列番号１に示す塩基配列において塩基番号306で終わる10〜30塩基長の塩基配列を有し、３’末端が蛍光色素で標識されている、または、配列番号２に示す塩基配列において塩基番号296から始まる10〜30塩基長の塩基配列を有し、５’末端が蛍光色素で標識されていることを特徴とする。 The probe of the present invention is a nucleic acid probe whose end is labeled with a fluorescent dye, and the fluorescence of the fluorescent dye decreases when hybridized, and has a length of 10 to 30 bases ending with base number 306 in the base sequence shown in SEQ ID NO: 1. It has a base sequence and is labeled with a fluorescent dye at the 3 ′ end, or has a base sequence of 10 to 30 bases starting from base number 296 in the base sequence shown in SEQ ID NO: 2, and the 5 ′ end is fluorescent It is characterized by being labeled with a dye.
本発明プローブは、配列番号１に示す塩基配列（G636A変異における正常型の塩基を有する配列）において配列番号１に示す塩基配列において塩基番号306で終わる10〜30塩基長の塩基配列、または、配列番号２に示す塩基配列において塩基番号296から始まる10〜30塩基長の塩基配列を有する他は、特許文献１に記載された消光プローブと同様でよい。本発明に使用される消光プローブの塩基配列の例としては、配列番号８または９に示すものが挙げられる。蛍光色素としては、特許文献１に記載されたものが使用できるが、具体
例としては、FAM(商標)、TAMRA(商標)、BODIPY(商標) FL等が挙げられる。蛍光色素のオリゴヌクレオチドへの結合方法は、通常の方法、例えば特許文献１に記載の方法に従って行うことができる。
The probe of the present invention is a base sequence having a length of 10 to 30 bases that ends with base number 306 in the base sequence shown in SEQ ID NO: 1 in the base sequence shown in SEQ ID NO: 1 (sequence having normal type base in G636A mutation) The base sequence shown in No. 2 may be the same as the quenching probe described in Patent Document 1 except that it has a base sequence of 10 to 30 bases starting from base No. 296. Examples of the base sequence of the quenching probe used in the present invention include those shown in SEQ ID NO: 8 or 9. As the fluorescent dye, those described in Patent Document 1 can be used, and specific examples include FAM (trademark), TAMRA (trademark), BODIPY (trademark) FL, and the like. The method for binding the fluorescent dye to the oligonucleotide can be performed according to a conventional method, for example, the method described in Patent Document 1.
本発明検出方法は、一塩基多型の部位を有する核酸について、蛍光色素で標識された核酸プローブを用いて、蛍光色素の蛍光を測定することにより融解曲線分析を行い、融解曲線分析の結果に基づいて変異を検出する方法であって、一塩基多型は、G636A変異であり、核酸プローブは本発明プローブであることを特徴とする。 In the detection method of the present invention, a melting curve analysis is performed on a nucleic acid having a single nucleotide polymorphism site by measuring the fluorescence of the fluorescent dye using a nucleic acid probe labeled with the fluorescent dye. A method for detecting a mutation based on the mutation, wherein the single nucleotide polymorphism is a G636A mutation, and the nucleic acid probe is a probe of the present invention.
本発明検出方法は、G636A変異を含む領域を増幅すること、および、本発明プローブを用いることの他は、通常の核酸増幅および融解曲線分析（Tm解析）の方法に従って行うことができる。 The detection method of the present invention can be carried out according to the usual methods of nucleic acid amplification and melting curve analysis (Tm analysis) other than amplifying a region containing the G636A mutation and using the probe of the present invention.
核酸増幅の方法としては、PCRポリメラーゼを用いる方法が好ましく、その例としては、PCR、ICAN、LAMP等が挙げられる。PCRポリメラーゼを用いる方法により増幅する場合は、本発明プローブの存在下で増幅を行うことが好ましい。用いるプローブに応じて、増幅の反応条件等を調整することは当業者であれば容易である。これにより、核酸の増幅後にプローブのTmを解析するだけなので、反応終了後増幅産物を取り扱う必要がない。よって、増幅産物による汚染の心配がない。また、増幅に必要な機器と同じ機器で検出することが可能なので、容器を移動する必要すらない。よって、自動化も容易である。 As a method for nucleic acid amplification, a method using PCR polymerase is preferred, and examples thereof include PCR, ICAN, LAMP and the like. When amplification is performed by a method using PCR polymerase, amplification is preferably performed in the presence of the probe of the present invention. It is easy for those skilled in the art to adjust the amplification reaction conditions and the like according to the probe used. As a result, only the Tm of the probe is analyzed after amplification of the nucleic acid, so that it is not necessary to handle the amplification product after the reaction is completed. Therefore, there is no worry of contamination by amplification products. Moreover, since it can detect with the same apparatus as an apparatus required for amplification, it is not necessary to move a container. Therefore, automation is also easy.
以下、PCRを用いる場合を例として、さらに説明する。PCRに用いるプライマー対は、本発明プローブがハイブリダイゼーションできる領域が増幅されるようにする他は、通常のPCRにおけるプライマー対の設定方法と同様にして設定することができる。プライマーの長さおよびTmは、通常には、12mer〜40merで40〜70℃、好ましくは16mer〜30merで55〜60℃である。プライマー対の各プライマーの長さは同一でなくてもよいが、両プライマーのTmはほぼ同一（通常には、相違が２℃以内）であることが好ましい。なお、Tm値は最近接塩基対(Nearest Neighbor)法により算出した値である。プライマー対の例としては、配列番号３および４に示す塩基配列を有するプライマーからなるものが挙げられる。 Hereinafter, the case where PCR is used will be further described as an example. The primer pair used for PCR can be set in the same manner as the primer pair setting method in ordinary PCR, except that the region where the probe of the present invention can hybridize is amplified. The length and Tm of the primer are usually 12 to 40 mer and 40 to 70 ° C., preferably 16 to 30 mer and 55 to 60 ° C. The length of each primer in the primer pair may not be the same, but the Tm of both primers is preferably substantially the same (usually, the difference is within 2 ° C.). The Tm value is a value calculated by the nearest base pair (Nearest Neighbor) method. Examples of primer pairs include those consisting of primers having the base sequences shown in SEQ ID NOs: 3 and 4.
PCRは、本発明で使用される本発明プローブの存在下で行うことが好ましい。これにより、増幅反応終了後に増幅産物を取り扱う操作を行うことなくTm解析を行うことができる。用いるプローブに応じて、プライマーのTmやPCRの反応条件を調整することは当業者であれば容易である。 PCR is preferably performed in the presence of the probe of the present invention used in the present invention. Thus, Tm analysis can be performed without performing an operation for handling the amplification product after the amplification reaction is completed. It is easy for those skilled in the art to adjust the Tm of the primer and the PCR reaction conditions according to the probe to be used.
代表的なＰＣＲ反応液の組成を挙げれば、以下の通りである。 A typical PCR reaction solution composition is as follows.
また、代表的な温度サイクルを挙げれば、以下の通りであり、この温度サイクルを通常25〜40回繰り返す。 Moreover, if a typical temperature cycle is mentioned, it will be as follows and this temperature cycle will be repeated 25-40 times normally.
(1) 変性、90〜98℃、1〜60秒
(2) アニーリング、50〜70℃、10〜60秒
(3) 伸長、60〜75℃、10〜180秒
(1) Denaturation, 90-98 ° C, 1-60 seconds
(2) Annealing, 50-70 ° C, 10-60 seconds
(3) Elongation, 60-75 ° C, 10-180 seconds
アニーリングおよび伸長を一ステップで行う場合には、50〜70℃、10〜180秒の条件が挙げられる。 In the case where annealing and elongation are performed in one step, conditions of 50 to 70 ° C. and 10 to 180 seconds can be mentioned.
Tm解析は、本発明プローブの蛍光色素の蛍光を測定する他は通常の方法に従って行うことができる。蛍光の測定は、蛍光色素に応じた波長の励起光を用い発光波長の光を測定することに行うことができる。Tm解析における昇温速度は、通常には、0.1〜1℃／秒である。Tm解析を行うときの反応液の組成は、プローブとその塩基配列に相補的な配列を有する核酸とのハイブリダイゼーションが可能であれば特に制限されないが、通常には、一価の陽イオン濃度が1.5〜5 mM、pHが７〜９である。PCR等のDNAポリメラーゼを用いる増幅方法の反応液は、通常、この条件を満たすので、増幅後の反応液をそのままTm解析に用いることができる。 The Tm analysis can be performed according to a usual method except that the fluorescence of the fluorescent dye of the probe of the present invention is measured. The fluorescence can be measured by measuring light having an emission wavelength using excitation light having a wavelength corresponding to the fluorescent dye. The rate of temperature increase in Tm analysis is usually 0.1-1 ° C./second. The composition of the reaction solution for performing Tm analysis is not particularly limited as long as hybridization between the probe and a nucleic acid having a sequence complementary to the base sequence is possible, but usually the monovalent cation concentration is 1.5-5 mM, pH is 7-9. Since the reaction solution of the amplification method using DNA polymerase such as PCR normally satisfies this condition, the amplified reaction solution can be used as it is for Tm analysis.
Tm解析の結果に基づくG636A変異の検出は通常の方法に従って行うことができる。本発明における検出とは、変異の有無の検出の他、変異型DNAの定量、正常型DNAと変異型DNAの割合の測定も包含する。 Detection of the G636A mutation based on the result of Tm analysis can be performed according to a usual method. The detection in the present invention includes not only detection of the presence or absence of mutation, but also quantification of mutant DNA and measurement of the ratio of normal DNA to mutant DNA.
本発明プローブを用いて、本発明の検出方法に用いるためのキットを提供することができる。このキットは、末端が蛍光色素で標識され、ハイブリダイゼーションしたときに蛍光色素の蛍光が減少する核酸プローブ（消光プローブ）であって、配列番号１に示す塩基配列において塩基番号306で終わる10〜30塩基長の塩基配列を有し、３’末端が蛍光色素で標識されている、または、配列番号２に示す塩基配列において塩基番号296から始まる10〜30塩基長の塩基配列を有し、５’末端が蛍光色素で標識されている核酸プローブを含むことを特徴とする。 A kit for use in the detection method of the present invention can be provided using the probe of the present invention. This kit is a nucleic acid probe (quenching probe) whose end is labeled with a fluorescent dye and the fluorescence of the fluorescent dye decreases when hybridized, and ends with base number 306 in the base sequence shown in SEQ ID NO: 1 It has a base sequence with a base length and is labeled with a fluorescent dye at the 3 ′ end, or has a base sequence with a length of 10 to 30 bases starting from base number 296 in the base sequence shown in SEQ ID NO: 2 and 5 ′ It comprises a nucleic acid probe whose end is labeled with a fluorescent dye.
この検出キットは、消光プローブの他に、本発明の検出方法における核酸増幅を行うのに必要とされる試薬類、特にDNAポリメラーゼを用いる増幅のためのプライマーをさらに含んでいてもよい。 In addition to the quenching probe, this detection kit may further contain reagents necessary for performing nucleic acid amplification in the detection method of the present invention, particularly primers for amplification using DNA polymerase.
この検出キットにおいて消光プローブ、プライマーおよびその他の試薬類は、別個に収容されていてもよいし、それらの一部が混合物とされていてもよい。 In this detection kit, the quenching probe, primer and other reagents may be accommodated separately, or a part of them may be a mixture.
以下に、本発明を実施例により具体的に説明する。 Hereinafter, the present invention will be specifically described by way of examples.
CYP2C19遺伝子のcDNAの636位のＡ→Ｇ変異（G636A変異）の部位を含む塩基配列（配列番号１、塩基番号302がCYP2C19遺伝子のcDNAの636位に相当）に基づき、G636A変異を含む部分を増幅できるように表２に示すプライマーを設計した。表２中、位置は、配列番号１に示す塩基配列における塩基番号を示す。 Based on the nucleotide sequence containing the site of the A → G mutation (G636A mutation) at position 636 of the cDNA of CYP2C19 gene (SEQ ID NO: 1, base number 302 corresponds to position 636 of the cDNA of CYP2C19 gene), the portion containing the G636A mutation The primers shown in Table 2 were designed so that they could be amplified. In Table 2, the position indicates the base number in the base sequence shown in SEQ ID NO: 1.
次に、表３に示す、末端部にCを有するプローブを設計した。表３中、位置は、配列番号１に示す塩基配列における塩基番号を示す。また、塩基配列中の大文字は、G636A変異の部位を示し、3'末端の(P)は、リン酸化されていることを示す。TAMRA(商標)による標識は、常法に従って行った。 Next, probes having C at the end shown in Table 3 were designed. In Table 3, the position indicates the base number in the base sequence shown in SEQ ID NO: 1. The capital letters in the base sequence indicate the G636A mutation site, and the (P) at the 3 ′ end indicates that it is phosphorylated. Labeling with TAMRA ™ was performed according to a conventional method.
精製ヒトゲノム（GFX Genomic Blood DNA Purification Kitダイレクト法にて全血から抽出）をサンプルとして、Smart Cycler System（Cephied)を用い、以下の条件でPCRおよびTm解析を行った。Tm解析における励起波長および検出波長は、それぞれ527〜555 nmおよび565〜605 nm（TAMRA）であった。 Using purified human genome (extracted from whole blood by GFX Genomic Blood DNA Purification Kit direct method) as a sample, PCR and Tm analysis were performed using Smart Cycler System (Cephied) under the following conditions. The excitation wavelength and detection wavelength in Tm analysis were 527 to 555 nm and 565 to 605 nm (TAMRA), respectively.
各プローブを用いてPCRおよびTm解析を行った結果、プローブ5T-mt-F1-18および3T-wt-F2-15を用いたときのみ、Tm解析で解析の可能な蛍光強度の変化が認められた。なお、各プローブのG636A変異を含む塩基配列に対する配置を図１および２に示す。図中、Wild配列およびmutant配列は、それぞれ配列番号１および２の塩基配列の塩基番号278〜317である。また、図中、Ｆは蛍光色素を示す。プローブがTm解析で使用できるかどうかは、蛍光色素を結合させたCの位置に依存すると考えられ、プローブの長さは、多型部位を含む限り、あまり重要でないと考えられる。 As a result of PCR and Tm analysis using each probe, only when probes 5T-mt-F1-18 and 3T-wt-F2-15 were used, changes in fluorescence intensity that can be analyzed by Tm analysis were observed. It was. In addition, the arrangement | positioning with respect to the base sequence containing G636A mutation of each probe is shown to FIG. In the figure, the Wild sequence and the mutant sequence are base numbers 278 to 317 of the base sequences of SEQ ID NOs: 1 and 2, respectively. In the figure, F represents a fluorescent dye. Whether the probe can be used in Tm analysis is considered to depend on the position of C to which the fluorescent dye is bound, and the length of the probe is considered to be less important as long as the polymorphic site is included.
サンプルとして、0.01μlの血液から抽出したＤＮＡ（３サンプル）ならびに変異型配列および正常型配列を有するDNA（それぞれ、mt/mtおよびwt/wt）ならびに変異型配列および正常型配列の両方を有するDNA（wt/mt）を調製し、プローブ3T-wt-F2-15を用いて定量を行った。結果を図３に示す。また、血液の量を変えて（0.01〜１μl）定量を行った結果を図４に示す。これらの結果から、この定量方法は再現性がよく、感度が高いことが分かる。プローブ5T-mt-F1-18を用いた場合も同様の結果が得られた。 As samples, DNA extracted from 0.01 μl of blood (3 samples) and DNA having mutant and normal sequences (mt / mt and wt / wt, respectively) and DNA having both mutant and normal sequences (Wt / mt) was prepared and quantified using probe 3T-wt-F2-15. The results are shown in FIG. Moreover, the result of having carried out fixed_quantity | quantitative_assay by changing the quantity of blood (0.01-1 microliter) is shown in FIG. From these results, it can be seen that this quantification method has good reproducibility and high sensitivity. Similar results were obtained when the probe 5T-mt-F1-18 was used.
なお、図３及び４において縦軸は、蛍光強度の一次導関数の逆符号の値（-dF/dt）、横軸は温度（℃）である。 3 and 4, the vertical axis represents the value of the inverse sign of the first derivative of fluorescence intensity (-dF / dt), and the horizontal axis represents temperature (° C).
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