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Hepatities E virus detection

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E型肝炎の検出システムの開発

Publicada em: Saúde e medicina
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Hepatities E virus detection

  1. 1. HEV検出システムの新規構築 http://www.tsukuba-genetech.com/ 1
  2. 2. E型肝炎 2 原因ウイルス: HEV 分 類: ヘペウイルス科 主な感染経路: 糞便で汚染された飲み水を介した糞口感染 潜 伏 期 間: 平均6週間 症 状 ・ 慢性化することがある。 ・ キャリア化することがある。 ・ 終生免疫が成立するか否かは不明。 ・ 急性肝炎を発症した場合はA型肝炎に類似。 ・ 稀に劇症化し死亡することあり。 ・ 妊婦がHEVに感染して発症した場合、劇症化する率が高い。 ・ 人獣共通感染症と認識されている唯一の肝炎ウイルスである。 診 断 ・血清学的検査: IgM抗体、IgA抗体の検出(まれに擬陽性、擬陰性がみられる) ・核酸増幅検査: HEV-RNAの検出(特殊な検査;一部の研究室で診断可能) 治 療 方 法: 急性期の対症療法 劇症化した場合は、血漿交換、人工肝補助療法、肝移植など
  3. 3. Genotype1 Genotype2 Genotype3 Genotype4 AvianHEV 3c 3b 3a 0.1 substitutions/site 0.1 Mex-14 I1 C4 C1 SAR-55 P1pSK-HEV-2 C3C5 P2 India TK15-92l I4I3HPESVPB1 T3 Morocco HE-JA2JAK-SaiHE-JI4swJ13-1JSF-Tot03CHE-JK4JYN-Nii02LJYN-Sap01CJSN-Sap-FHJSN-Sap-FH02CHE-JA1HE-JF3 JSW-Sap95JKK-SapHE-JF5HE-JF4JYW-Sap02JTS-Sap02HE-JA19HE-JA37HE-JA41HE-JA36HE-JA28 CCC220 E087-SAP04C SH-SW-zs1 E067-SIJ05C swCH31 IND-SW-00-01 swCH25 T1 swGX32 JKO-ChiSai98C Ch-S-1 JYI-ChiSai01C HEVN2 swGX40 swDQ Avian Osh205swMN06-C1056 swX07-E1 E116-YKH98C HE-JA04-1911swJ12-4 swJ8-5swMN06-A1288 E088-STM04C Arkell HE-JA10JMY-Haw JKN-Sap HEV-US1HEV-US2 swMengpSHEV-3 pSHEV-2pSHEV-1 wbJYG1 swJ570 E097-OSA05C JRA1 JBOAR1-Hyo04JMO-Hyo03L JSO-Hyo03LJYO-Hyo03LJTH-Hyo03LJDEER-Hyo03LJJT-Kan HEVN1 wbJSG1 wbJTS1 JMNG-Oki02CJE03-1760F p5B JE03-1760F p5 JE03-1760F p13-3 JE03-1760F p10JE03-1760F 118d-AlexJE03-1760F p0JE03-1760F 114-A549JE03-1760F wt HEV遺伝子ORF2塩基配列による分子系統樹 (Genoype1~4が明確に分離できる) 2008年12月の段階で、DNA data bankに登録されているE型肝炎ウイルスのORF2遺伝子全てを解析し、 HEVのウイルス遺伝子をRealtimeRT-PCRで特異的に検出できるprimer set (HEV Universal RealtimeRT- PCR primer)、Genotype 3およびGenotype 4 を特異的に検出できるprimer set (HEV Genotype3/4 detection primer)を考案、設計した。 HEV検出システム HEV Universal RealtimeRT-PCR primer set ↓ HEV Genotype3/4 detection primer set 左図の、発現しているHEV遺伝子を全て検出でき、 さらにGenotype3およびGenotype4の遺伝子を特 異的に検出・同定できる。 人獣共通感染 E型肝炎ウイルス (HEV)検出システム
  4. 4. 4 HEV universal primer 設計例1:forward side HEVuniF83 Geno- type III Geno- type I II and IV
  5. 5. 5 HEVG3F151 Geno- type III Geno- type I II and IV HEV universal primer 設計例2:forward side Frd-18
  6. 6. 6 HEVuniR197 HEVuniR223 HEV universal primer 設計例3:reverse side Geno- type III Geno- type I II and IV Rsv-18
  7. 7. 7 PrimerのPosition (HEV ORF2 region: ca 2000nt) 431 HEVuniF83 HEVuniR197 HEVuniR223 HEVG3F151 HEVG3R323 HEVG4F150 HEVG4R388 G4 G1,G2,G3 (Note: primer nameの数字はprimerの5’-endのpositionを示す。) Primer作成) 1) Genotype I~IVまでuniversalに増幅できるprimerを約120bp付近で設計した。 設計参考配列:E型肝炎ウイルス98種類 Genotype I~II 18種類, Genotype III 42種類, Genotype IV 38種類 2) Primer regionは全てのgenotypeの保存領域とした。 3) ForwardおよびReverse primer sequenceのBLAST解析を実施し、Forwardおよび Reverse ともに全てHEVであった。 *主なプライマーを表示した(青色)。 4)上記プライマーを参考に、PCR産物が100bp以下になるよう再設計(Frd-18, Rsv-18)し、 BLAST解析で全てのHEVを認識することを確認した。 Frd-18 Rsv-18
  8. 8. HEV検出系 H28年版 方法 I. 抽出 ブタ糞 ↓ 約80-100mg/ 800-1000ml in PBS ↓ 撹拌, 1分 ↓ 遠心, 10Krpm 30秒 ↓ 上清 ↓ QuickGene RNA tissue kit SII 100ml II. RT-PCR RT反応 ↓ HEVRT Rsv18 primer: xxxxxxxxxxxxx * ポジコン用HEV-RNA断片 condition) 65C 60min, 70C 5min, 4C hold PCR反応 ↓ Frd18 primer:xxxxxxxxxxx Tag primer:ttttttttttt condition) 95C 9min (1cycle), 95C 30sec 66C 30sec 72C 30sec (28cycles), 72C 5min (1cycle) 電気泳動 HEVRT Rvs-18 5’ 3’ 5’ 3’ Frd-18 primer Tag primer 抽出されたHEVRT RNAに対し、HEVRT Rvs-18 primerでcDNAを合成する。 原理 cDNA cDNAに対し、Frd-18 primerとTag primerで増幅し95bp断片を検出する。 RNA
  9. 9. 電気泳動 2.5% agarose gel, 5ml PCR product A1, A2: ブタ糞 Aから個々に抽出(1,2) B1, B2: ブタ糞 Bから個々に抽出(1,2) DW1: RT反応におけるDW(ネガティブコントロール) DW2: PCR反応におけるDW(ネガティブコントロール) P: HEV-RNA断片(ポジティブコントロール) M DW2 DW1 A1 A2 B1 B2 A1+P A2+P B1+P B2+P M 500bp 200bp
  10. 10. 10 【検出感度】 1: ポジコン用HEV-RNA断片 (101 molecukes/ ml) 2: ポジコン用HEV-RNA断片 (102 molecukes/ ml) 3: ポジコン用HEV-RNA断片 (103 molecukes/ ml) 4: ポジコン用HEV-RNA断片 (104 molecukes/ ml) 5: ポジコン用HEV-RNA断片 (106 molecukes/ ml) 6: ポジコン用HEV-RNA断片 (101 molecukes/ ml) 7: ポジコン用HEV-RNA断片 (102 molecukes/ ml) 8: ポジコン用HEV-RNA断片 (103 molecukes/ ml) 9: ポジコン用HEV-RNA断片 (104 molecukes/ ml) 10: ポジコン用HEV-RNA断片 (106 molecukes/ ml) 11: DW (1st PCR, nega-con) 12: DW (2nd PCR, nega-con) 1 ~ 5: 090216に調製したRT用サンプル 6~10: 090514に調製したRT用サンプル *090527にすべてのサンプルをRT反応した。 1 2 3 4 5 6 7 8 9 10 M 11 12 ・ポジコン用HEV-RNAのロット、保管期間などにより検出感度が異なるが、 電気泳動では10分子以上を検出可能である。 100bp
  11. 11. 11 PAS (PrintedArrayStrip) E型肝炎ウイルスのRNAの検出 1: control (no virus RNA) 2: HEV RNA + 10分子 3: HEV RNA + 1分子 1 2 3 II. RT-PCR RT反応 ↓ HEVRT Rsv18 primer condition) 65C 60min, 70C 5min, 4C hold PCR反応 ↓ [F-1] Frd18 primer Biotin Tag primer condition) 95C 9min (1cycle), 95C 30sec 66C 30sec 72C 30sec (28cycles), 72C 5min (1cycle) PAS反応 ↓ 【展開】 PCR 産物 + 展開液 ↓ 【染色】青色染色アビジンビーズ反応 ↓ 【乾燥】
  12. 12. 12 【結果】 ウイルス98種類をすべて、一セットのプイラマーで検出するの に、適した領域をin silicoで検討した結果、ORF2の中の一領域 が適していることが判った。PCR産物の大きさ70塩基で、大きさ が小さいことから、PCRに必要な時間は少なくすることが出来た。 既存のウイルスRNAから、当該領域を含む520塩基を調製し、 各実験の鋳型として、検出感度を求めた。その結果、検出感度 は10分子程度であることが判った。 【考察】 現時点では、E型肝炎ウイルス98種類については、検出できる ことは判っているが、今後、データベースに登録されている他の ウイルスが検出できるか、また、今後登録されるウイルスについ ても、同様にin silicoで検討して行く予定である。

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