JP2006180884A - C型肝炎ウイルスの検出のための試薬および方法 - Google Patents
C型肝炎ウイルスの検出のための試薬および方法 Download PDFInfo
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Abstract
【解決手段】オリゴヌクレオチドプライマーを含む構成物はcDNAを作製するためのウイルスゲノムRNAの逆転写およびそれに続くポリメラーゼ連鎖反応によるcDNAの増幅を含む方法において、C型肝炎ウイルスの核酸を増幅し、検出するために使用される。オリゴヌクレオチドプローブはハイブリダイゼーションによる増幅されたDNAの存在の検出に使用される。C型肝炎ウイルスの核酸の存在を検出するキットに有用である。
【選択図】図1A
Description
から選択される部分配列を含む。
の一つを使用するために上流プライマーの次のグループ:
から選択される核酸配列を含む。
が含まれる。本発明はさらに非C9−HCVの配列を検出するためのプローブを提供するものである。
C9 29 pHCV−C9 75265 1992年7月2日
R110 32 pHCV−R110 75266 1992年7月2日
50%グリセロール 16.00
10xRT反応緩衝液(100mM Tris−HCl
(pH8.3)、900mM塩化カリウム) 10.00
dGTP(10mM);終濃度200μM 2.00
dATP(10mM);終濃度200μM 2.00
dUTP(10mM);終濃度200μM 2.00
dCTP(10mM);終濃度200μM 2.00
KY80(配列番号5);15μM(最終反応あたり
15pmole);ビオチン化(+)鎖プライマー 1.00
KY78(配列番号18);15μM(最終反応あたり
15pmole);ビオチン化(−)鎖RTプライマー 1.00
UNG:1単位/μl 2.00
rTh DNAポリメラーゼ:2.5単位/μl(1x
酵素保存緩衝液*) 4.00
MnCl2(10mM);終濃度0.9mM 9.00
チューブあたりのマスター混合液 50.00
RNA(キャリア**とともに水に溶解) 50.00
全反応容量 100.00
1.カバーを予め熱するためにTC9600サーモサイクラーをオンにする。
2.室温で反応液を準備する。
3.サーモサイクラーにチューブを入れ、サーモサイクラーを再始動するために「運転」を押す。
4.ファイル8で機械を始動する。
5.提案されたサーモサイクラーの条件:
ファイル1:50℃で2分間保持(UNG弱化段階)
ファイル2:70℃で15分間保持(逆転写段階)
ファイル3:95℃で1分間保持
ファイル4:二段階温度、95℃15秒および60℃10から30秒を二回
ファイル5:二段階温度、90℃15秒および60℃10から30秒を38回
ファイル6:72℃で1時間保持
ファイル7:15℃に保持、無制限
ファイル8:ファイル1、2、3、4、5、6および7を連結
Claims (29)
- C型肝炎ウイルス核酸の存在を検出するためのオリゴヌクレオチドプローブを含む構成物で、前記プローブはJapan、USおよびC9の原型株からなるグループから選択されるHCV株または変異株の核酸を検出するのに適している構成物。
- 前記プローブがJapan、USおよびC9のHCV株を検出するのに適している請求項1に記載の構成物。
- 前記プローブが、KY67(配列番号10)、KY78(配列番号18)、KY81(配列番号11)、KY86(配列番号13)、KY95(配列番号20)、KY150(配列番号43)およびKY145(配列番号19)とその相補的な配列からなる核酸成分のグループから選択される配列内に含まれる少なくとも14ヌクレオチドとハイブリダイズする核酸部分配列を含む請求項2に記載の構成物。
- 前記プローブがJapanおよびUSのHCV原型ウイルスの核酸を検出するのに適している請求項1に記載の構成物で、前記プローブはKY83(配列番号7)、KY87(配列番号14)、KY84(配列番号8)、KY88(配列番号12)、KY85(配列番号9)、KY100(配列番号21)、KY148(配列番号17)、KY149(配列番号16)、KY65(配列番号1)、KY68(配列番号27)、KY82(配列番号28)、KY99(配列番号26)、KY109(配列番号24)、KY111(配列番号25)、KY80(配列番号5)、KY144(配列番号6)およびKY153(配列番号15)とその相補的な配列からなるグループから選択される配列内に含まれる少なくとも14ヌクレオチドにハイブリダイズする核酸部分配列を含む構成物。
- 前記プローブがJapanおよびUSのHCV原型ウイルスの核酸を検出するのに適しており、さらに前記プローブがHCV−C9原型ウイルスの核酸を検出しない請求項1に記載の構成物。
- 請求項5に記載の構成物で、前記プローブはKY83(配列番号7)、KY87(配列番号14)、KY84(配列番号8)、KY88(配列番号12)、KY85(配列番号9)、KY100(配列番号21)、KY148(配列番号17)およびKY149(配列番号16)とその相補的な配列からなるグループから選択される配列内に含まれる少なくとも14ヌクレオチドとハイブリダイズする核酸部分配列を含む構成物。
- 前記プローブがHCV−C9原型ウイルスの核酸を検出するのに適しており、さらに前記プローブがJapanおよびUSのHCV原型ウイルスの核酸を検出しない請求項1に記載の構成物。
- 前記プローブが配列番号34、配列番号35、配列番号36および配列番号37とその相補的な配列からなるグループから選択される配列内に含まれる少なくとも14ヌクレオチドにハイブリダイズする核酸部分配列を含む請求項7に記載の構成物。
- HCVの核酸を増幅するためのオリゴヌクレオチドプライマーを含む構成物で、前記プライマーはJapan、USおよびC9株からなるグループから選択されるHCV株または同型の核酸部分配列を増幅するのに適している構成物。
- 前記プライマーがJapan、USおよびC9のHCV株を増幅するのに適している請求項9に記載の構成物。
- 請求項10に記載の構成物で、前記プライマーがKY67(配列番号10)、KY78(配列番号18)、KY80(配列番号5)、KY81(配列番号11)、KY83(配列番号7)、KY86(配列番号13)、KY88(配列番号12)、KY95(配列番号20)、KY100(配列番号21)、KY144(配列番号6)、KY153(配列番号15)およびKY145(配列番号19)からなるグループから選択される核酸部分配列を含む構成物。
- 請求項9に記載の構成物で、前記プライマーがJapanおよびUSのHCV原型ウイルスの核酸を増幅するのに適しており、さらにKY65(配列番号1)、KY68(配列番号27)、KY98(配列番号3)、KY99(配列番号26)、KY109(配列番号24)、KY111(配列番号25)およびKY149(配列番号16)からなるグループから選択される核酸部分配列を含む構成物。
- 前記プライマーがJapanおよびUSのHCV原型ウイルスの核酸を増幅するのに適しており、さらにHCV−C9原型ウイルスの核酸は増幅しない請求項9に記載の構成物。
- 前記プライマーがKY87(配列番号14)、KY84(配列番号8)、KY148(配列番号17)およびKY85(配列番号9)からなるグループから選択される核酸部分配列を含む請求項13に記載の構成物。
- 前記プライマーがHCV−C9原型ウイルスの核酸を増幅するのに適しており、さらにJapanおよびUSのHCV原型ウイルスの核酸を増幅しない請求項9に記載の構成物。
- 前記プライマーが配列番号38、配列番号39、配列番号40、配列番号41および配列番号42からなるグループから選択される核酸部分配列を含む請求項15に記載の構成物。
- C型肝炎のゲノム核酸の存在を検出する方法であって、前記C型肝炎のゲノム核酸はJapan、USおよびC9原型株からなるグループから選択される核酸であって、
(a) 核酸部分配列を増幅すること;
(b) プローブが核酸部分配列に結合し、安定な雑種二重鎖を形成するような条件下で、増幅された核酸をその部分配列に特異的なオリゴヌクレオチドプローブと混合すること;および
(c) その部分配列およびプローブの間に形成される雑種を検出すること;
からなる方法。 - 肝炎ウイルスのC9単離株を検出する方法であって、その方法は
(a) HCV−C9の核酸配列を含むことが予想される試料をHCV−C9の核酸配列に相補的な部分配列をもつ核酸プローブと接触させること;および
(b) プローブの前記配列とのハイブリダイゼーションを検出すること;
の段階からなる方法。 - 請求項18に記載の方法であって、さらに段階(a)の前に、前記HCV−C9の核酸配列の部分配列を増幅する段階を含む方法。
- 請求項17または請求項18に記載の方法であって、請求項1に記載のプローブが使用される方法。
- 請求項17または請求項19に記載の方法であって、増幅が請求項9に記載の少なくとも一つのプライマーを使用して行われる方法。
- 請求項17または請求項19に記載の方法であって、増幅がポリメラーゼ連鎖反応により行われる方法。
- C型肝炎ウイルスの核酸の存在を検出するためのキットであって、Japan、USおよびC9原型株からなるグループから選択されるHCV株または変異株の核酸を検出するのに適している請求項1に記載の少なくとも一つの核酸プローブを含むキット。
- C型肝炎ウイルスのC9単離株に特異的な核酸を検出するための請求項23に記載のキットであって、HCV−C9ウイルスの核酸部分配列に実質的に結合する少なくとも一つの核酸プローブを含むキット。
- C型肝炎ウイルスのC9単離株に特異的な核酸を検出するための請求項23に記載のキットであって、請求項7に記載の少なくとも一つの核酸プローブを含むキット。
- 配列番号29またはその変異種と実質的に同一である核酸配列を含む単離されたC型肝炎ウイルス。
- 変異核酸配列がR116(配列番号30)、R45(配列番号31)、R110(配列番号32)またはR43(配列番号33)と実質的に同一である請求項26に記載の単離されたC型肝炎ウイルス。
- ATCC寄託番号第75265号の単離されたC型肝炎ウイルス。
- ATCC寄託番号第75266号の単離されたC型肝炎ウイルス。
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US75130591A | 1991-08-27 | 1991-08-27 | |
US91884492A | 1992-07-21 | 1992-07-21 |
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JP2006042214A Expired - Lifetime JP4164098B2 (ja) | 1991-08-27 | 2006-02-20 | C型肝炎ウイルスの検出のための試薬および方法 |
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US (2) | US5527669A (ja) |
EP (2) | EP0529493B1 (ja) |
JP (2) | JP3790274B2 (ja) |
KR (1) | KR100280758B1 (ja) |
CN (2) | CN1047630C (ja) |
AT (2) | ATE241704T1 (ja) |
AU (1) | AU665952B2 (ja) |
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