JP4194147B2 - TT virus genotyping method - Google Patents
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Description
【０００１】
【発明の属する技術分野】
本発明は、ＴＴウイルス( 以下、「ＴＴＶ」 ということがある）の遺伝子型分類方法に関する。本発明は、ＴＴＶの感染経路の解明や、病態の解明等に有用である。
【０００２】
【従来の技術】
ＴＴＶは、輸血後肝炎を起こすウイルスとして、１９９７年、Nishizawa らによって発見された新たな肝炎ウイルスである(Okamoto,H., Nishizawa,T., Kato,N., Ukita,M., Ikeda,H., Iizuka,H., Miyakawa,Y. and Mayumi,M., “Molecular cloning and characterization of a novel DNA virus(TTV) associated with posttransfusion hepatitis of unknown etiology", JOURNAL Hepatol. Res. 10, 1-16 (1998)) 。このウイルスは、エンベロープを持たないDNA ウイルスで、糞便中に排泄され経口感染を起こすことが示唆されその感染率は比較的高いことが予測される。
【０００３】
一般に、ウイルスは変異を比較的起こしやすい。遺伝子を検出することが本質的であるが、遺伝子を検出する場合重要となるのが、その配列情報である。なぜなら、変異の存在する部分に不適切なプライマーやプローブを設定すると一般に、検出率が低下する。したがって、正確な診断には、より多くの遺伝情報が必要であり、また、より適切なプライマーやプローブの設定と測定系の開発が必要とされる。
【０００４】
ウイルスを分類する場合、遺伝子の相同性％を用いる場合があるがこの方法は、正確でない。最近の、分子生物学の進歩によって、いろいろな生物種間で遺伝子配列を比較分類する場合、塩基置換数から、系統樹を用いて系統的にウイルスを分類することが可能となった。しかしながら、そのためには、それぞれの配列を比較しalignment を作製し、配列間の塩基置換数を正確に推定することが必要である。塩基配列を比較し塩基置換数を推定する場合、一番大切なことは、多重置換が生じることである。たとえば、２つの遺伝子配列X 、Z のある対応する配列がG とT であったとすると、共通祖先の塩基、たとえば、A から、G(X)→ C→T(Z)となったのか、A から、G(X)→A →C →T(Z)という順番に多重置換したあとにT(Z)となったのか不明確である。この多重置換は相同性％のみの解析では補正できない。多重置換を補正する方法は現在は6-paramater 法が最も正確である。さらに、Bootstrap 解析を行うことで最も正確な系統樹を作製でき、正確なウイルスの分類を可能とする。ウイルスの分類は感染経路の解明や、病態の解明などに有用な技術である。
【０００５】
ＴＴＶは最近発見された新しいウイルスであるため、多数の株の遺伝子配列が知られているわけではなく、従って、その遺伝子型分類方法も提案されていない。特に、上記したBootstrap 解析に基づく分類方法は、全く知られていない。
【０００６】
【発明が解決しようとする課題】
従って、本発明の目的は、ＴＴＶの遺伝子型について、Bootstrap 解析に基づいて正確に分類することができる、ＴＴＶの遺伝子型の分類方法を提供することである。
【０００７】
【課題を解決するための手段】
本願発明者らは、３００例ものＴＴＶ検体を世界から集め、その特定の領域をＰＣＲにより増幅して塩基配列を決定し、それらをBootstrap 解析に基づいて厳密に比較することにより、ＴＴＶの遺伝子型を正確に分類する方法を考え出し、本発明を完成した。
【０００８】
すなわち、本発明は、ＴＴウイルスの遺伝子型を分類する方法であって、ＴＴウイルスの1984nt〜1987ntに位置するMesI部位、2001nt〜2006ntに位置する第１のNdeI部位、2014nt〜2019ntに位置する第２のNdeI部位、2033nt〜2038ntに位置する第１のPstI部位、2066nt〜2069ntに位置するNalIII部位及び／又は2072nt〜2077ntに位置する第２のPstI部位の有無を調べ、
(1) 前記 MesI 部位、第２の NdeI 部位及び NalIII 部位を有し、前記した他の制限酵素部位を有さないもの、
(2) 前記第２の NdeI 部位及び NalIII 部位を有し、前記した他の制限酵素部位を有さないもの、
(3) 前記第１の PstI 部位を有し、前記第２の PstI 部位を有するか又は有さず、前記した他の制限酵素部位を有さないもの、
(4) 前記 MesI 部位並びに第１及び第２の PstI 部位を有し、前記した他の制限酵素部位を有さないもの、
(5) 前記 NalIII 部位を有し、前記した他の制限酵素部位を有さないもの、
(6) 前記第１の NdeI 部位並びに前記第１及び第２の PstI 部位を有し、前記した他の制限酵素部位を有さないもの、
(7) 前記した制限酵素部位を１つも有さないもの
の７つの遺伝子型のいずれかに分類することを含む、ＴＴウイルスの遺伝子型分類方法を提供する。
【０００９】
【発明の実施の形態】
上記のように、本発明の方法では、ＴＴＶの1984nt〜1987ntに位置するMesI部位、2001nt〜2006ntに位置する第１のNdeI部位、2014nt〜2019ntに位置する第２のNdeI部位、2033nt〜2038ntに位置する第１のPstI部位、2066nt〜2069ntに位置するNalIII部位及び／又は2072nt〜2077ntに位置する第２のPstI部位の有無に基づいてＴＴＶの遺伝子型を分類する。なお、上記した各塩基番号( 「nt」で表示）は、国立遺伝学研究所の遺伝子データベースDDBJにAccession No. AB008394で登録されている配列の5'末端を1nt として数えたものである。なお、MesI部位の塩基配列はTTAAであり、NdeI部位の塩基配列はCATATGであり、PstI部位の塩基配列はCTGCAGであり、NalIII部位の塩基配列はCATGである。
【００１０】
本発明の方法に従って分類される１つの遺伝子型( 本明細書では「G1a 型」 と呼ぶ）は、前記MesI部位、第２のNdeI部位及びNalIII部位を有し、前記した他の制限酵素部位を有さないものである。本発明の方法に従って分類される他の１つの遺伝子型( 本明細書では「G1b 型」 と呼ぶ）は、前記第２のNdeI部位及びNalIII部位を有し、前記した他の制限酵素部位を有さないものである。本発明の方法に従って分類される他の１つの遺伝子型( 本明細書では「G2型」 と呼ぶ）は、前記第１のPstI部位を有し、前記第２のPstI部位を有するか又は有さず、前記した他の制限酵素部位を有さないものである。本発明の方法に従って分類される他の１つの遺伝子型( 本明細書では「G3型」 と呼ぶ）は、前記MesI部位並びに第１及び第２のPstI部位を有し、前記した他の制限酵素部位を有さないものである。本発明の方法に従って分類される他の１つの遺伝子型( 本明細書では「G4型」 と呼ぶ）は、前記NalIII部位を有し、前記した他の制限酵素部位を有さないものである。本発明の方法に従って分類される他の１つの遺伝子型( 本明細書では「G5型」 と呼ぶ）は、前記第１のNdeI部位並びに前記第１及び第２のPstI部位を有し、前記した他の制限酵素部位を有さないものである。本発明の方法に従って分類される他の１つの遺伝子型( 本明細書では「G6型」 と呼ぶ）は、前記した制限酵素部位を１つも有さないものである。
【００１１】
上記した各型とその制限酵素部位の有無を表にまとめると下記表１に示す通りである。
【００１２】
【表１】
−：認識せず
【００１３】
上記した分類法を組み合わせることにより、ＴＴＶをG1a 、G1b 、G2、G3、G4、G5及びG6の７つの型に分類することができる。もっとも、本発明は、ＴＴＶを必ずしも上記７つの型に分類する方法に限定されるものではなく、上記７つの型のいずれか１つとそれ以外、又は上記の７つの型のうち任意の複数の型とそれらに含まれない型とに分類する方法等も本発明の範囲に含まれる。
【００１４】
本発明の分類方法は、上記６箇所の制限酵素部位の有無に基づくものであるので、上記４種類の制限酵素を用いたＲＦＬＰ( 制限酵素断片長多型) 法により好都合に行なうことができる。ＲＦＬＰ法は、制限酵素で消化後の核酸断片を電気泳動により分離し、臭化エチジウム等で染色してバンドの位置を調べ、その電気泳動パターンによって制限酵素部位の有無を調べる方法である。ＲＦＬＰ法自体はこの分野において周知である。
【００１５】
さらに、ＲＦＬＰを行なう前にＰＣＲ等の核酸増幅法により、調べるべき領域を増幅しておくことが感度の点から好ましい。ＰＣＲ自体は周知であり、そのためのキット及び装置も市販されている。なお、上記のようにＴＴＶ遺伝子の基本的な塩基配列は公知であるので、本発明の方法において調べるべき領域を含む領域、好ましくは1912nt〜2186ntの領域を含む領域を増幅するようにプライマーを設定して容易にＰＣＲを行なうことができる。また、ＰＣＲは２回行ない、最初のＰＣＲで増幅された断片内の領域を２回目のＰＣＲで増幅することにより(nested PCR)、ＰＣＲの信頼性をより高めることができる。なお、ＰＣＲに用いるプライマーの例は下記実施例に記載されている。
【００１６】
なお、本発明の方法は、上記の制限酵素部位の有無を調べればよいので、必ずしもＲＦＬＰ法により行なう必要はない。例えば、1912nt〜2186ntの全塩基配列を調べ、その配列に基づいて上記の制限酵素部位の有無を調べることも可能である。
【００１７】
ＲＦＬＰ法により行なう場合、例えば次のように行なうと効率良く分類を行なうことができる。すなわち、核酸増幅法によりＴＴＶ遺伝子の調べるべき領域を増幅後の検体を先ずNdeI及びPstIでそれぞれ消化してＲＦＬＰ法を行なう。その電気泳動パターンよりG2及びG5は分類を特定することができる。G2及びG5以外の型である場合、G1a 及びG1b か、これら以外の型かはその電気泳動パターンから知ることができる。G1a かG1b であった場合には、次いで別途MesIで消化してＲＦＬＰ法を行なうことにより、G1a とG1b とを分類することができる。一方、NdeI及びPstIでそれぞれ消化してＲＦＬＰ法を行なった結果、G4、G6又はG3であった場合には、さらに別途NalIII及びMseIでそれぞれ消化し、ＲＦＬＰ法を行なうことにより、G4、G6及びG3を分類することができる。
【００１８】
【実施例】
以下、本発明を実施例に基づきさらに具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。
【００１９】
実施例１
(1) TTV DNA の抽出
日本人、供血者、慢性肝炎患者合計300 例を用いてＴＴＶの検出を行った。
血清0.2 ｍｌから、High Pure Viral Nucleic Acid Kit (製品名； Boehringer Mannheim) を用いて抽出し、DNA 溶液５０μl を得た。
【００２０】
(2) ＰＣＲ
DNA 溶液25μl と配列番号２及び３のプライマーをそれぞれ10pmol用いてPCR を行った。条件は、変性95℃30秒、アニール60℃、４５秒、伸長72℃、４５秒で、合計50μl とし、1.25U EXTaq （商品名；宝酒造）を使用し、３５サイクルのＰＣＲを行った。さらに、ひきつづき、配列番号4 及び5 のプライマーをそれぞれ10pmol用いてPCR を行った。条件は、変性95℃30秒、アニール60℃、４５秒、伸長72℃、４５秒で、合計50μl とし、1.25U EXTaq （商品名；宝酒造）を使用し、３５サイクルのＰＣＲを行った。増幅産物は、電気泳動によって確認した。陽性率は、５１％であった。
【００２１】
陽性であった増幅産物は、マイクロスピンカラムＳ400 （商品名；ファルマシア）を用いて精製し、配列番号６、７のプライマー、BigDye terminator cycle sequencing kit（商品名；パーキンエルマー）、ABI377 autosequencer（商品名；パーキンエルマー）を用いてその塩基配列を決定した。決定された塩基配列を配列番号８以下に示す。なお、上記のＰＣＲにより増幅された領域は、ＴＴＶの1912nt〜2186ntの領域である。なお、配列番号８以下には、増幅領域から両端のプライマー部分を除いた1939nt〜2160ntの塩基配列を示す。
【００２２】
(3) 分子系統解析
得られた塩基配列を最も相同性の高くなるように並べ、６- パラメーター法（gojobori, et.al., J Mol Evol, 18, 414-423, 1982)により、遺伝学的距離を推定し、NJ methods (Saitou and Nei, Mol Biol Evol, 4, 406-425 ,1987)で系統解析を行った。さらに、最も信頼性の高い系統樹を得るために、bootstrap resampling tests( Felsenstein, Evolution, 39, 783-791, 1985)を1000回行った。その結果、上記のように７つの遺伝子型に分類することにより最も信頼性の高い系統樹が得られた。BOOTSTRAP 解析によって85.7％以上で他のTYPEと系統樹が分かれる事をあきらかにした（系統樹）。得られた系統樹を図１に示す。
【００２３】
実施例２
(1) ＰＣＲ
日本人５５人から実施例1 と同様にして調製したDNA 溶液25μl と配列番号２及び３のプライマーをそれぞれ10pmol用いてPCR を行った。条件は、変性95℃30秒、アニール60℃、４５秒、伸長72℃、４５秒で、合計50μl とし、1.25U EXTaq （商品名；宝酒造）を使用し、３５サイクルのＰＣＲを行った。さらに、ひきつづき、配列番号１及び３のプライマーをそれぞれ10pmol用いてPCR を行った。条件は、変性95℃30秒、アニール60℃、４５秒、伸長72℃、４５秒で、合計50μl とし、1.25U EXTaq （商品名；宝酒造）を使用し、３５サイクルのＰＣＲを行った。増幅産物は、電気泳動によって確認した。陽性率は、５１％であった。なお、上記のＰＣＲにより増幅された領域は、ＴＴＶの1912nt〜2186ntの領域である。
【００２４】
(2) ＲＦＬＰ
それぞれの制限酵素で切断後、電気泳動パターンによって型判定を行った。すなわち、ＰＣＲ産物２μl を、それぞれ、１０U の( 商品名；New England Biolabs) (商品名；New England Biolabs)それぞれの添付バッファーを用いて、３７℃、 (商品名；New England Biolabs)で、６５℃で３時間反応させ、0.5 μｇ/ ｍｌ、エチジウムブロマイドの３％アガロースゲルで電気泳動後、紫外線照射下で写真撮影を行い、上記のとうり検出バンドで各タイプに判定、分類した。
【００２５】
すなわち、上記方法により各制限酵素消化により得られるバンドの長さは下記表２に示す通りである。
【００２６】
【表２】
【００２７】
上記の方法により、未知の検体を分類する場合に、次のように行なうと効率が良いのでこの方法によった。すなわち、まず、配列番号１及び３のプライマーによるＰＣＲ増幅産物をNdeI, PstIでそれぞれ処理し、NdeIで消化して105+170bp ならG1a 又はG1b 、91+184bpならＧ５( 確定）、PstIで消化して127+148 bpならＧ２( 確定) である。これらのバンドが検出されない場合、さらに、新にNalIII, MseIで切断する。NalIIIで消化して159+116 bpならＧ４( 確定) 、275 bpならＧ６( 確定）、MseIで消化して73+202bpならG3( 確定）、275bp ならＧ２( 確定）である。また、上記のNdeI, PstI消化でG1a, G1bと判定された場合、MseIで新に切断し、73+202bpならG1a(確定）、275bp ならG1b(確定）と判定する。
【００２８】
(3) 結果
日本人５５例では、G1a 型 10例、G1b 型17例、G2型19例,G1a+1b 型、5 例、G3型、１例、G4型 １例、G5型 １例、G6型 １例であった。なお、G1a+1b型は、G1a 型及びG1b 型の２種類のウイルスに感染していた例である。
【００２９】
【発明の効果】
本発明により、ＴＴＶの遺伝子型を正確に分類する方法が提供された。本発明は、ＴＴＶの感染経路の解明や、病態の解明等に役立つ。
【００３０】
【配列表】
【図面の簡単な説明】
【図１】実施例１で作成された系統樹を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for genotyping a TT virus (hereinafter sometimes referred to as “TTV”). The present invention is useful for elucidating the infection route of TTV, elucidating the disease state, and the like.
[0002]
[Prior art]
TTV is a new hepatitis virus discovered by Nishizawa et al. In 1997 as a virus causing post-transfusion hepatitis (Okamoto, H., Nishizawa, T., Kato, N., Ukita, M., Ikeda, H ., Iizuka, H., Miyakawa, Y. And Mayumi, M., “Molecular cloning and characterization of a novel DNA virus (TTV) associated with posttransfusion hepatitis of unknown etiology”, JOURNAL Hepatol. Res. 10, 1-16 ( 1998)). This virus is a non-enveloped DNA virus that is excreted in the stool and causes oral infection, and its infection rate is expected to be relatively high.
[0003]
In general, viruses are relatively susceptible to mutation. It is essential to detect a gene, but the sequence information is important when detecting a gene. This is because if an inappropriate primer or probe is set in a portion where a mutation exists, the detection rate generally decreases. Therefore, more genetic information is required for accurate diagnosis, and more appropriate primer and probe settings and measurement systems need to be developed.
[0004]
When classifying viruses,% gene homology may be used, but this method is not accurate. Recent advances in molecular biology have made it possible to systematically classify viruses using a phylogenetic tree based on the number of base substitutions when comparing and classifying gene sequences among various species. However, in order to do so, it is necessary to compare each sequence and create an alignment to accurately estimate the number of base substitutions between sequences. When estimating the number of base substitutions by comparing base sequences, the most important thing is that multiple substitution occurs. For example, if the corresponding sequences of two gene sequences X and Z are G and T, is the common ancestor base, for example, A changed from G (X) → C → T (Z)? Therefore, it is unclear whether T (Z) is obtained after multiple permutation in the order of G (X) → A → C → T (Z). This multiple substitution cannot be corrected by analysis of only% homology. The 6-paramater method is currently the most accurate method for correcting multiple permutations. Furthermore, the most accurate phylogenetic tree can be created by performing Bootstrap analysis, enabling accurate virus classification. Virus classification is a useful technique for elucidating the route of infection and elucidating disease states.
[0005]
Since TTV is a recently discovered new virus, the gene sequences of many strains are not known, and therefore no genotyping method has been proposed. In particular, there is no known classification method based on the Bootstrap analysis described above.
[0006]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a TTV genotype classification method that can accurately classify TTV genotypes based on Bootstrap analysis.
[0007]
[Means for Solving the Problems]
The present inventors collected as many as 300 TTV specimens from the world, amplified specific regions thereof by PCR, determined the nucleotide sequences, and strictly compared them based on Bootstrap analysis, thereby obtaining the TTV genotype. The present invention has been completed by devising a method for accurately classifying the above.
[0008]
That is, the present invention is a method for classifying a TT virus genotype, which comprises a MesI site located at 1984 nt to 1987 nt, a first NdeI site located at 2001 nt to 2006 nt, and a first NdeI site located at 2014 nt to 2019 nt. The presence or absence of two NdeI sites, a first PstI site located at 2033 nt to 2038 nt, a NalIII site located at 2066 nt to 2069 nt and / or a second PstI site located at 2072 nt to 2077 nt ,
(1) the MesI site, a second NdeI site and NalIII site, having no other restriction enzyme sites described above,
(2) having the second NdeI site and NalIII site and not having the other restriction enzyme sites described above,
(3) having said first PstI site, said or no having a second PstI site, having no other restriction enzyme sites described above,
(4) has the MesI portion and the first and second PstI site, having no other restriction enzyme sites described above,
(5) having said NalIII site, having no other restriction enzyme sites described above,
(6) having the first NdeI site and the first and second PstI sites and not having the other restriction enzyme sites described above,
(7) Those having no restriction enzyme site
A method of genotyping a TT virus comprising classifying the genotype into any one of the seven genotypes of:
[0009]
DETAILED DESCRIPTION OF THE INVENTION
As described above, in the method of the present invention, the MesI site located at 1984 nt to 1987 nt, the first NdeI site located at 2001 nt to 2006 nt, the second NdeI site located at 2014 nt to 2019 nt, 2033 nt to 2038 nt The TTV genotype is classified based on the presence or absence of the first PstI site located, the NalIII site located 2066 nt to 2069 nt, and / or the second PstI site located 2072 nt to 2077 nt. Each of the above base numbers (indicated by “nt”) is obtained by counting the 5 ′ end of the sequence registered as Accession No. AB008394 in the gene database DDBJ of the National Institute of Genetics as 1 nt. The base sequence of the MesI site is TTAA, the base sequence of the NdeI site is CATATG, the base sequence of the PstI site is CTGCAG, and the base sequence of the NalIII site is CATG.
[0010]
One genotype classified according to the method of the present invention (referred to herein as “G1a type”) has the MesI site, the second NdeI site, and the NalIII site, and the other restriction enzyme sites described above. It does not have. Another genotype (referred to herein as “G1b type”) classified according to the method of the present invention has the second NdeI site and NalIII site, and has the other restriction enzyme sites described above. It is not to be. One other genotype (referred to herein as “G2 type”) classified according to the method of the present invention has the first PstI site and has or has the second PstI site. It does not have the other restriction enzyme sites described above. Another genotype classified according to the method of the present invention (referred to herein as “G3 type”) has the MesI site and the first and second PstI sites, and the other restriction enzymes described above. It has no part. Another genotype classified according to the method of the present invention (referred to herein as “G4 type”) has the NalIII site and does not have the other restriction enzyme sites described above. Another genotype classified according to the method of the present invention (referred to herein as "G5 type") has the first NdeI site and the first and second PstI sites, as described above. It does not have other restriction enzyme sites. Another genotype classified according to the method of the present invention (referred to herein as “G6 type”) is one that does not have any of the aforementioned restriction enzyme sites.
[0011]
Table 1 below summarizes each type and the presence or absence of its restriction enzyme site.
[0012]
[Table 1]
By combining the above classification methods, TTVs can be classified into seven types: G1a, G1b, G2, G3, G4, G5 and G6. However, the present invention is not necessarily limited to the method of classifying the TTV into the above seven types, and any one of the above seven types and the others, or any plurality of the above seven types. And a method for classifying them into types not included in them are also included in the scope of the present invention.
[0014]
Since the classification method of the present invention is based on the presence or absence of the above six restriction enzyme sites, it can be conveniently performed by the RFLP (Restriction Enzyme Fragment Length Polymorphism) method using the above four types of restriction enzymes. The RFLP method is a method in which a nucleic acid fragment digested with a restriction enzyme is separated by electrophoresis, stained with ethidium bromide or the like, the position of the band is examined, and the presence or absence of a restriction enzyme site is examined by the electrophoresis pattern. The RFLP method itself is well known in the art.
[0015]
Furthermore, it is preferable from the viewpoint of sensitivity that the region to be examined is amplified by a nucleic acid amplification method such as PCR before performing RFLP. PCR itself is well known, and kits and devices for it are also commercially available. Since the basic nucleotide sequence of the TTV gene is known as described above, a primer is set so as to amplify a region including a region to be examined in the method of the present invention, preferably a region including a region of 1912nt to 2186nt. Thus, PCR can be easily performed. Further, PCR is performed twice, and the region in the fragment amplified by the first PCR is amplified by the second PCR (nested PCR), whereby the reliability of the PCR can be further increased. Examples of primers used for PCR are described in the following examples.
[0016]
Note that the method of the present invention is not necessarily performed by the RFLP method because the presence or absence of the above-mentioned restriction enzyme site may be examined. For example, it is possible to examine the entire base sequence from 1912nt to 2186nt and examine the presence or absence of the above restriction enzyme site based on the sequence.
[0017]
When the RFLP method is used, for example, the following classification can be performed efficiently. That is, the sample after amplification of the region to be examined for the TTV gene by the nucleic acid amplification method is first digested with NdeI and PstI, respectively, and the RFLP method is performed. The classification of G2 and G5 can be specified from the electrophoresis pattern. In the case of a type other than G2 and G5, whether it is G1a and G1b or other types can be known from the electrophoresis pattern. In the case of G1a or G1b, G1a and G1b can be classified by separately digesting with MesI and performing the RFLP method. On the other hand, as a result of digesting with NdeI and PstI and performing RFLP, respectively, if G4, G6 or G3, further digesting with NalIII and MseI and performing RFLP separately, G4, G6 and G3 can be classified.
[0018]
【Example】
Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
[0019]
Example 1
(1) Extraction of TTV DNA TTV was detected using a total of 300 Japanese, blood donors, and chronic hepatitis patients.
Extracted from 0.2 ml of serum using High Pure Viral Nucleic Acid Kit (product name: Boehringer Mannheim) to obtain 50 μl of DNA solution.
[0020]
(2) PCR
PCR was performed using 25 μl of DNA solution and 10 pmol of each of the primers of SEQ ID NOs: 2 and 3. The conditions were denaturation 95 ° C. 30 seconds, annealing 60 ° C., 45 seconds, extension 72 ° C., 45 seconds, a total of 50 μl, and 35 cycles of PCR were performed using 1.25 U EXTaq (trade name; Takara Shuzo). Subsequently, PCR was performed using 10 pmol each of the primers of SEQ ID NOs: 4 and 5. The conditions were denaturation 95 ° C. 30 seconds, annealing 60 ° C., 45 seconds, extension 72 ° C., 45 seconds, a total of 50 μl, and 35 cycles of PCR were performed using 1.25 U EXTaq (trade name; Takara Shuzo). The amplification product was confirmed by electrophoresis. The positive rate was 51%.
[0021]
The positive amplification product was purified using a microspin column S400 (trade name: Pharmacia), the primers of SEQ ID NOs: 6 and 7, BigDye terminator cycle sequencing kit (trade name; Perkin Elmer), ABI377 autosequencer (trade name) The base sequence was determined using Perkin Elmer). The determined nucleotide sequence is shown in SEQ ID NO: 8 and below. In addition, the region amplified by the above PCR is a region of 1912 nt to 2186 nt of TTV. SEQ ID NO: 8 and below show a base sequence of 1939 nt to 2160 nt excluding the primer portions at both ends from the amplification region.
[0022]
(3) Molecular phylogenetic analysis The obtained base sequences are arranged so as to have the highest homology, and genetics are determined by the 6-parameter method (gojobori, et.al., J Mol Evol, 18, 414-423, 1982). The phylogenetic analysis was performed using NJ methods (Saitou and Nei, Mol Biol Evol, 4, 406-425, 1987). Furthermore, bootstrap resampling tests (Felsenstein, Evolution, 39, 783-791, 1985) were performed 1000 times to obtain the most reliable phylogenetic tree. As a result, the most reliable phylogenetic tree was obtained by classifying into 7 genotypes as described above. BOOTSTRAP analysis revealed that other TYPEs and phylogenetic trees were separated by more than 85.7% (phylogenetic tree). The obtained phylogenetic tree is shown in FIG.
[0023]
Example 2
(1) PCR
PCR was carried out using 25 μl of a DNA solution prepared in the same manner as in Example 1 from 55 Japanese and 10 pmol of the primers of SEQ ID NOs: 2 and 3. The conditions were denaturation 95 ° C. 30 seconds, annealing 60 ° C., 45 seconds, extension 72 ° C., 45 seconds, a total of 50 μl, and 35 cycles of PCR were performed using 1.25 U EXTaq (trade name; Takara Shuzo). Subsequently, PCR was performed using 10 pmol of each of the primers of SEQ ID NOs: 1 and 3. The conditions were denaturation 95 ° C. 30 seconds, annealing 60 ° C., 45 seconds, extension 72 ° C., 45 seconds, a total of 50 μl, and 35 cycles of PCR were performed using 1.25 U EXTaq (trade name; Takara Shuzo). The amplification product was confirmed by electrophoresis. The positive rate was 51%. In addition, the region amplified by the above PCR is a region of 1912 nt to 2186 nt of TTV.
[0024]
(2) RFLP
After digestion with each restriction enzyme, the type was determined based on the electrophoresis pattern. That is, 2 μl of the PCR product was added to each of 10 U (trade name; New England Biolabs) (trade name; New England Biolabs) at 37 ° C. (trade name; New England Biolabs) at 65 ° C. The mixture was reacted for 3 hours, electrophoresed on a 3% agarose gel of 0.5 μg / ml ethidium bromide, photographed under ultraviolet irradiation, and determined and classified into each type by the above-mentioned band detection band.
[0025]
That is, the length of the band obtained by digesting each restriction enzyme by the above method is as shown in Table 2 below.
[0026]
[Table 2]
[0027]
When an unknown specimen is classified by the above method, the following method is used because it is efficient if performed as follows. That is, first, PCR amplification products using the primers of SEQ ID NOs: 1 and 3 were treated with NdeI and PstI, respectively, digested with NdeI and digested with G1a or G1b for 105 + 170 bp, G5 (confirmed) for 91 + 184 bp, and PstI. If it is 127 + 148 bp, it is G2 (confirmed). If these bands are not detected, they are further cleaved with NalIII and MseI. Digested with NalIII is G4 (confirmed) if 159 + 116 bp, G6 (confirmed) if 275 bp, G3 (confirmed) if digested with MseI and 73 + 202 bp, G2 (confirmed) if 275 bp. If G1a or G1b is determined by digestion with NdeI or PstI as described above, it is newly cleaved with MseI. If 73 + 202 bp, G1a (determined) is determined, and if 275 bp, G1b (determined) is determined.
[0028]
(3) Results In 55 Japanese cases, G1a type 10 cases, G1b type 17 cases, G2 type 19 cases, G1a + 1b type 5 cases, G3 type 1 case, G4 type 1 case, G5 type 1 case, G6 There was one type. The G1a + 1b type is an example in which two types of viruses, G1a type and G1b type, were infected.
[0029]
【The invention's effect】
The present invention provides a method for accurately classifying TTV genotypes. The present invention is useful for elucidating the infection route of TTV, elucidating the disease state, and the like.
[0030]
[Sequence Listing]
[Brief description of the drawings]
FIG. 1 is a diagram showing a phylogenetic tree created in Example 1. FIG.
Claims (3)
(1) 前記 MesI 部位、第２の NdeI 部位及び NalIII 部位を有し、前記した他の制限酵素部位を有さないもの、
(2) 前記第２の NdeI 部位及び NalIII 部位を有し、前記した他の制限酵素部位を有さないもの、
(3) 前記第１の PstI 部位を有し、前記第２の PstI 部位を有するか又は有さず、前記した他の制限酵素部位を有さないもの、
(4) 前記 MesI 部位並びに第１及び第２の PstI 部位を有し、前記した他の制限酵素部位を有さないもの、
(5) 前記 NalIII 部位を有し、前記した他の制限酵素部位を有さないもの、
(6) 前記第１の NdeI 部位並びに前記第１及び第２の PstI 部位を有し、前記した他の制限酵素部位を有さないもの、
(7) 前記した制限酵素部位を１つも有さないもの
の７つの遺伝子型のいずれかに分類することを含む、ＴＴウイルスの遺伝子型分類方法。 A method for classifying TT virus genotypes , comprising a MesI site located between 1984 nt and 1987 nt of a TT virus, a first NdeI site located between 2001 nt and 2006 nt, a second NdeI site located between 2014 nt and 2019 nt, 2033 nt The presence or absence of a first PstI site located at ˜2038 nt, a NalIII site located at 2066 nt to 2069 nt and / or a second PstI site located at 2072 nt to 2077 nt ,
(1) the MesI site, a second NdeI site and NalIII site, having no other restriction enzyme sites described above,
(2) having the second NdeI site and NalIII site and not having the other restriction enzyme sites described above,
(3) having said first PstI site, said or no having a second PstI site, having no other restriction enzyme sites described above,
(4) has the MesI portion and the first and second PstI site, having no other restriction enzyme sites described above,
(5) having said NalIII site, having no other restriction enzyme sites described above,
(6) having the first NdeI site and the first and second PstI sites and not having the other restriction enzyme sites described above,
(7) Those having no restriction enzyme site
A method for genotyping a TT virus, comprising classifying the genotype into any one of the seven genotypes .
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