鳥インフルエンザ遺伝子検査法の特性と電気泳動による リアルタイム
Contents
1. HA AIV rPCR rPCR rPCR cPCR rPCR rPCR rPCR rPCR 572 f rPCR2. ees 68 569 574 2015 HPAD 1 9 2010 2011 9 24 H5N1 W 9 183 FEED H55L H7 AIV H5 H7 AIV AIV
3. H5 H7 cPCR 1step H5 H7 cPCR rPCR cPCR 1step 1step 2 cPCR RNA ED 10 2step 2step 1step 100 PCR Tm
4. RNA HA 10 11 HA PCR cPCR 12 14 PCR rPCR 15 19 19 21 rPCR 20 21
5. 571 68 569 574 2015 PCR 4 0 1 2 3 4 5 6 3 cPCR F 145 7 67 68 3 0 0 0 0 12 R 145 141 4 0 0 0 0 0 0 NP F 145 142 3 0 0 0 0 0 0 rS R 145 141 4 0 0 0 0 0 0 Es P 145 140 5 0 0 0 0 0 0 cPCR A F HP 224 198 25 1 0 0 0 0 0 12 R HP 224 217 7 0 0 0 0 0 0 cPCR B F HP 224 106 116 2 0 0 0 0 82 22 R HP 224 204 20 0 0 0 0 0 0 F HP 224 160 61 3 0 0 0 0 0 is R HP 224 159 64 1 0 0 0 0 0 P HP 224 210 14 0 0 0 0 0 0 cPCR A F LP 29 5 1 20 3 0 0 0 0 12 R LP 29 23 6 0 0 0 0 0 0 cPCR B F LP 29 1 0 20 6 0 1 1 5 22 R LP 29 0 25 2 2 0 0 0 0 F LP 29 5 21 1 1 1 0 0 0 mT R LP 29 95 4 0 0 0 0 0 0 E P LP 29 24 5 0 0 0 0 0 0 1 F R P 2 HP LP 3 5 rPCR rPCR m rPCR rPCR 0 1 2 3 4 5 6 NP Chiba H3 H5 Tsukuba H5 7 H7 Shimane H7 7 H7 N
6. 4 5 6 Chiba H3 A duck Tsukuba 9 2005 H5N2 Tsu kuba H5 A duck Shimane 83 2006 H7N3 Shi mane H7 A chicken NY 119055 7 2001 H7N2 NY H7 HA fii 4 096 RNA QIAamp Viral RNA Mini amp QIAGEN RNA 10 10 10 L Tsukamoto 19 rPCR Lee 5 12 cPCR rPCR rPCR PrimeScript RT reagent Kit Perfect Real Time EA325430 RNA 5x7 w 107 cDNA rPCR Premix Ex Taq Per fect Real Time 2 274 48 cDNA lul 20 7 95C 30 95C 10 50C 20 60C 32 35 cPCR 1
7. rPCR HPAI Chiba H3 H x m 1 Mase M Tsukamoto K Imada T Imai K Tanimura N Nakamura K Yamamoto Y Hitomi T Kira T Nakai T Kiso M Horimoto T Kawaoka Y Yamaguchi S Char acterization of H5N1 influenza A viruses isolated dur ing the 2003 2004 influenza outbreaks in Japan Virol ogy 332 167 176 2005 2 REA 42
8. RT PCR QIAGEN OneStep RT PCR Kit QIAGEN 1 cPCR lstep PrimeScript 1st strand cDNA Syn thesis Kit Z HFA T cPCR TaKaRa Ex Taq Z 2 2234 X8 2 cPCR 2step Prime Script 1st strand cDNA Synthesis Kit B cPCCR SapphireAmp Fast PCR Master Mix 93724308 2 cPCR 2step 3 1step NRNA 1z 2077 50C 30 95C 10 94C 30 45C 30 72C 45 35 72C 10 2step RNA 1x 10 cDNA cDNA lul 20 7 94C 10 94C 30 50C 30 72C 1 68 569 574 2015 35 72 10 2step 2step
9. cDNA cDNA 1z7 20z 94C 2 98C 5 50C 5 72C 10 35 72C 10 1 rPCR 2 rPCR ABI75000 X 3 15 Threshold 0 05 KARO 2 Threshold F A 18 145 H5 224 29 NCBI X2 rPCR NP H5 19 cPCR NP H5 12 cPCR H5 22
10. rPCR cPCR rPCR rPCR rPCR AIV 4 A duck Chiba 3 2007 H3N2 T T 854 0063 3118 120957 25 1331 FAX 0957 25 1332 E mail inoued00 pref nagasaki lg jp 569 68 569 574 2015 PCR X1 rPCR cPCR 1step 2step 2step F R p F R Chiba H3 NP NP 1200F 09 NP 1529R 0 NP Probe 0 19 NP 1200 0 NP 1529 0 12 Tsukuba H5 HA H5 H5 918F 1 H5 1166R 0 H5 Probe 0 19 H5 155f 3 H5 699r 0 12 Shimane H7 HA EuH7 H7 937Feu 0 H7 1177R 0 H7 Probe 0 19 H7 12f 7 H7 645r 0 12 NY H7 HA AmH7 H7 937Fau 0 H7 1177R 0 H7 Probe 0 19 H7 12f 6 H7 645r 0 12 1 EuH7 H7 2 AmH7 H7 3
11. EE SERIMIER PR RARER mp x PCR EX Cr 854 0063 3118 AAA XX 2014 10 31 2015 4E 5 18 PCR rPCR PCR cPCR rPCR cPCR 10 10 000 NP H5 rPCR rPCR rPCR
12. 1 4 2006 3 43 1 7 2007 4 PEZA 7 43 89 96 2007 5 Okamatsu M Saito T Yamamoto Y Mase M Tsuduku S Nakamura K Tsukamoto K Yamaguchi S Low pathogenicity H5N2 avian influenza outbreak in Japan during the 2005 2006 Vet Microbiol 124 35 46 2007 6 Sugiura K Yamamoto M Nishida T Tsukamoto D Saito T Onodera T Recent outbreaks of avian influ enza in Japan Rev Sci Tech 28 1005 1013 2009 7 45 23 31 2009 8 2009 H7N6 45 33 35 2009 9 Sakoda Y Ito H Uchida Y Okamatsu M Yamamoto N Soda K Nomura N Kuribayashi S Shichinohe S Sunden Y Umemura T Usui T Ozaki H Yamaguchi T Murase T Ito T Saito T Takada A Kida H Reintro duction of H5N1 highly pathogenic avian influenza virus by migrat
13. MEGA5 3 1 H5 1 9 H7 rPCR rPCR rPCR E rPCR 1step 2step 2step 570 2 3 EU HE um cPCR EINI 2004 Ee ERE FR as TES RR lstep 2step ios H3N2 3 200 7 MAS NP Chiba H3 4
14. 3 2 2 ee E v aL RR T H5 Tsukuba H5 4 2 ND 0 Ne T DES 7 Shimane H7 4 3 0 0 HAN2 1 200 BN EUN EN Eu HAN6 2 200 200 D 10 2 ND H5N1 109 1999 2012 AUR iude poe E ie9 2000 4 H5N2 8 HE NP rPCR 1 HENS uo x 2 2002 20008 A cPCR 3 H5 TIENI SaN ENN 2 1999 2008 Mi Tubes rPCR cPCR 2 0 rPCR 4 cPCR 6 H6N2 1 2006 55 a ri a o ME PUR RET E RU H5 cPCR 3 POM E S PEN rPCR OE UA rPCR HE X rPCR H9N2 1 2001 EM ee MORIA EIN E LL NUN C OMEN rPCR NN o 0 va NS nn 1 MNO LAE C INFE i PR 365 NP 10 1 r
15. 49 59 63 2013 96 123 2006 68 569 574 2015 PCR Comparison of Avian Influenzavirus Genetic Detection Assays and Verification of Real time PCR Results by Electrophoresis Daisuke INOUE Fumihiko YOSHINO Yoichi MUKOHARA and Shigeru IWAMATSU Chuo Livestock Hygiene Service Center Nagasaki Prefecture 3118 Kaizu Isahaya 854 0063 Japan SUMMARY The sensitivity of genetic detection assays of avian influenzavirus AIV was compared to show that real time polymerase chain reaction rPCR was 10 to 10 000 times more sensitive than conventional PCR cPCR Nucleotide sequences of the primers and probes for AIV genetic detection assays were compared with influenzavirus sequences registered to GenBank A few mismatches between the nucleotide sequences of the primers and probes for the assays targeting the NP gene and those of the strains were observed Several mismatches were observed in a case study targeting H5 genes raising concern that sensitivity might be lower against several low pathogenic strains Eelectrophoresis of an rPCR product was used for evaluation of the results showing that it is rapid and sensitive enough to discriminate non specific reaction caused in the rPCR assay Key words Avian Influe
16. J Vet Diagn Invest 19 625 634 2007 Monne I Ormelli S Salviato A De Battisti C Bettini F Salomoni A Drago A Zecchin B Capua I Cattoli G Development and validation of a one step real time PCR assay for simultaneous detection of subtype H5 H7 and H9 avian influenza viruses J Clin Microbiol 46 1769 1773 2008 Sidoti F Rizzo F Costa C Astegiano S Curtoni A Mandola ML Cavallo R Bergallo M Development of real time RT PCR assays for detection of type A influ enza virus and for subtyping of avian H5 and H7 hem agglutinin subtypes Mol Biotechnol 44 41 50 2010 Spackman E Senne DA Bulaga LL Trock S Suarez DL Development of multiplex real time RT PCR as a diagnostic tool for avian influenza Avian Dis 47 1087 1090 2003 Tsukamoto K Noguchi D Suzuki K Shishido M Ashizawa T Kim MC Lee YJ Tada T Broad detec tion of diverse H5 and H7 hemagglutinin genes of avian influenza viruses by real time reverse transcrip tion PCR using primer and probe sets containing mixed bases J Clin Microbiol 48 4275 4278 2010 Arafa AS Selim AA Hassan MK Aly MM Genetic characterization of variant strains of highly pathogen ic avian influenza H5NI that escaped detection by real time reverse transcriptase PCR diagnostic tests Avian Dis 54 673 676 2010 H5 KU H7 PCR
17. PCR S l NU RE NS nnn HINGS COD 11992 RR ka e rPCR 148 lstep 231 2step 216 2step 160 HONI 7228 3 oo A xd x z TS TEM ET 5 HSN RM AIV ORIS T BHL Ov CH ETTOIUKOR BE 0 i rPCR cPCR H5N2 20 2004 2008 i 21 FN 1976 2000 AIV rPCR MEE ME 3 x 1 OIE HA Avian Influenza Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 436 454 2013 3 rPCR C cPCR 10 10 000 f
18. Y H7 D 10 t 2 2 5 NP rPCR AIV H5 2 D 3 H5 cPCR 3 DNA 3 DNA DNA DNA H5 68 569 574 2015
19. nza Electrophoresis Genetic Detection Assay Mismatch Sensitivity T Correspondence to Daisuke INOUE Chuo Livestock Hygiene Service Center Nagasaki Prefecture 3118 Kaizu Isahaya 854 0063 Japan TEL 0957 25 1331 FAX 0957 25 1332 E mail inoued00Qpref nagasahi lg jb J Jpn Vet Med Assoc 68 569 574 2015 68 569 574 2015 574
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