JP6756835B2 - ウイルス性出血性敗血症ウイルスの遺伝子変異の検出方法 - Google Patents
ウイルス性出血性敗血症ウイルスの遺伝子変異の検出方法 Download PDFInfo
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Description
(b)温度を変化させながら上記ハイブリダイズされた産物と上記のPNAプローブ間の融解温度を得る段階;
(c)上記ハイブリダイズされた産物の融解温度をとそれぞれのPNAプローブに対して各々区間化して、各区間にコードを付与する段階;及び
(d)変異が予想される試料を上記(a)段階ないし(b)段階と同一の条件で反応させて融解温度を取得し、当該融解温度を、上記(c)段階によって付与されたコードに従ってコード化し、ウイルス性出血性敗血症ウイルス(VHSV)のNV遺伝子の変異の類型を判別する段階を含むレポーター(reporter)及び消光子(quencher)が結合されたPNAプローブを利用してウイルス性出血性敗血症ウイルス(VHSV)のNV遺伝子の変異を判別する方法を提供する。
(b)温度を変化させながら上記ハイブリダイズされた産物の融解温度を得る段階;
(c)上記ハイブリダイズされた産物の融解温度を、それぞれのPNAプローブに対して各々区間化し、各区間にてコードを付与する段階;及び
(d)変異が予想される試料を上記(a)段階ないし(b)段階と同一の条件で反応させて融解温度を取得し、当該融解温度を、上記(c)段階によって付与されたコードに従ってコード化し、これにより、ウイルス性出血性敗血症ウイルス(VHSV)のNV遺伝子の変異の類型を判別する段階を含むレポーター(reporter)及び消光子(quencher)が結合されたPNAプローブを利用してウイルス性出血性敗血症ウイルス(VHSV)のNV遺伝子の変異を判別する方法に関するものである。
[実施例]
以下、実施例を通じて本発明をより詳細に説明する。これらの実施例は単に本発明を例示するためのもので、本発明の範囲がこれらの実施例により制限されることで解釈されないことは当業界で通常の知識を有する者において自明である。
国立水産科学院で保管中であるVHSを起こしたウィルスの試料を利用してcDNA鋳型に塩基配列を解析してOIE(国際貿易事務局)のガイドラインに告示された塩基配列との一致の可否を確認し、保管中である試料の中でVHSVであることが確認された試料を使用した。
VHSVはRNA virusでcDNAで作製して実験に使用しており、RNAの抽出はTRIzol(登録商標) RNA Isolation Reagents(Thermo社、米国)を利用し、cDNAはM−MLV Reverse Transcriptase(enzynomics社、韓国)を利用して実験に使用した。
実施例2を利用した増幅曲線の結果を図10に示した。遺伝子増幅の可否を確認することができて、蛍光別にsampleが増幅されていることを確認することができる。
実施例2及び実施例3の結果を基に実際、VHSV資料のNV遺伝子変異の検出できる判定表を作ることができて、これを利用してNV遺伝子の変異検出が可能である。
Claims (7)
- 次の段階を含むレポーター(reporter)及び消光子(quencher)が結合されたPNAプローブを利用してウイルス性出血性敗血症ウイルス(VHSV)の非ビリオン(NV)遺伝子の変異を判別する方法:
(a)
(i)ウイルス性出血性敗血症ウイルス(VHSV)または上記ウイルス性出血性敗血症ウイルス(VHSV)のNV遺伝子の変異の部位を包含する標的核酸を含む試料及び
(ii)上記ウイルス性出血性敗血症ウイルス(VHSV)のNV遺伝子の変異の部位を含む標的核酸の塩基配列に相補的に結合し、配列番号1〜6の何れかひとつの配列で表示されるPNAプローブのひとつ以上
を混合して各PNAプローブを試料にハイブリダイズさせる段階;
(b)温度を変化させながら上記ハイブリダイズされた産物の融解温度を得る段階;
(c)上記ハイブリダイズされた産物の融解温度を、それぞれのPNAプローブに対して各々区間化し、各区間にコードを付与する段階;及び
(d)変異が予想される試料を上記(a)段階ないし(b)段階と同一の条件で反応させて変異融解温度を取得し、当該融解温度を、上記(c)段階によって付与されたコードに従ってコード化し、これにより、ウイルス性出血性敗血症ウイルス(VHSV)のNV遺伝子の変異の類型を判別する段階。 - 前記野生型の標的核酸とPNAプローブの間に付与されたコードと、変異型の標的核酸とPNAプローブの間に付与されたコードは異なることを特徴とする、請求項1に記載の方法。
- 前記レポーターはFAM(6−カルボキシフルオレセイン)、テキサスレッド(Texas red)、HEX(2’,4’,5’,7’,−テトラクロロ−6−カルボキシ−4,7−ジクロロフルオレセイン)及びCy5で構成される群から選択される一つ以上であることを特徴とする、請求項1に記載の方法。
- 前記消光子はTAMRA(6−カルボキシテトラメチル−ローダミン)、BHQ1、BHQ2及びDabcylで構成される群から選択される一つ以上であることを特徴とする、請求項1に記載の方法。
- 前記PNAプローブは配列番号1〜3で表示されるPNAプローブのセットまたは配列番号4〜6で表示されるPNAプローブのセットであることを特徴とする、請求項1に記載の方法。
- それぞれレポーター(reporter)及び消光子(quencher)が結合されており、ウイルス性出血性敗血症ウイルス(VHSV)のNV遺伝子に相補的に結合し、配列番号1〜6の何れかひとつの配列で表示されるPNAプローブのひとつ以上を含む、ウイルス性出血性敗血症ウイルス(VHSV)のNV遺伝子の変異された塩基配列の位置の確認用キット。
- 前記PNAプローブは配列番号1〜3で表示されるPNAプローブのセットまたは配列番号4〜6で表示されるPNAプローブのセットであることを特徴とする、請求項6に記載のキット。
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