Klassische und real-time RT-PCR-Teste

Transkript

1 F L I Klassische und real-time RT-PCR-Teste - molekulare Diagnostik aviärer Influenzavirusinfektionen -! "!"#$ % &!'(!!'(! #"#)* +,!"#$!!'(!!'(!-.( /0 * /" *

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3 .,9 #"&6 '(!& & & 9;.#. 6 +,9 &!'(!@,& )' ) & "( 00? 0? A1 A? #' Lfd-Nr Val-Nr Isolat HA-type NA-type 1 H1-1 A/Fort Mammoth/1/47 H1 N1 2 H2-1 A/duck/Potsdam/177/83 H2 N2 3 H2-2 A/guinea fowl/germany/dz3/85 H2 N2 4 H2-3 A/Mallard/Germ/Wv K/03 H2 5 H3-1 A/duck/Ukraine/1/63 H3 N8 6 H3-2 A/Mallard/Germ/Wv K/03 H3 N8 7 H4-1 A/duck/Czechoslowakia/56 H4 N6 8 H4-2 A/Mallard/Germ/Wv K/03 H4 9 H5-1 A/tern/S.Africa/61 H5 N3 10 H5-2 A/chicken/Scotland/59 H5 N1 11 H5-3 A/duck/Potsdam/2216/84 H5 N6 12 H5-4 A/duck/Potsdam/2243/84 H5 N6 13 H5-5 A/duck/Potsdam/617/85 H5 N2 14 H5-6 A/duck/Potsdam/619/85 H5 N2 15 H5-7 A/duck/Potsdam/1701/85 H5 N2 16 H5-8 A/duck/Potsdam/1402/86 H5 N2 17 H5-9 A/duck/Potsdam/1403/86 H5 N2 18 H5-10 A/chicken/Italy/8/98 H5 N2 19 H5-11 A/chicken/Italy/22/98 H5 N9 20 H5-12 A/Hongkong/156/97 H5 N1 21 H5-13 A/chicken/Indonesia/R /03 H5 N1 22 H5-14 A/mallard/Germany/Wv K/03 H5 N2 23 H5-15 A/mallard/Germany/Wv K/03 H5 N2?N3?N9? 24 H5-16 A/teal/Germany/Wv K/03 H5 N2 25 H5-17 A/mallard/Germany/Wv474-77K/04 H5 N? 26 H5-18 A/chicken/Vietnam/P41/05 H5 N1 27 H5-19 A/chicken/Vietnam/P78/05 H5 N1 28 H5-20 A/chicken/Vietnam/P22/05 H5 N1 29 H5-21 A/duck/Vietnam/TG36-H2/05 H5 N1 30 H5-22 A/duck/Vietnam/AG40-O2/05 H5 N1 31 H5-23 A/duck/Vietnam/TG24-O1/05 H5 N1 32 H5-24 A/chicken/GXLA/1204/05 H5 N1 33 H5-25 A/chicken/Indonesia/R132/1/04 H5 N1 34 H5-26 A/chicken/Indonesia/R132/2/04 H5 N1 35 H5-27 A/chicken/Indonesia/R132/3/04 H5 N1 36 H5-28 A/chicken/Indonesia/R60/05 H5 N1 37 H6-1 A/turkey/Mass/3740/65 H6 N2 38 H6-2 A/turkey/Grub/R41/98 H6 N5 39 H6-3 A/turkey/Kartzfehn/R26/99 H6 N2 Lfd-Nr Val-Nr Isolat HA-type NA-type 40 H6-4 A/turkey/Heidemark/R30/99 H6 N1 41 H6-5 A/turkey/Poeppel/R04/02 H6 N2 42 H7-1 A/chicken/Brescia/1902 H7 N1 43 H7-2 A/FPV/dutch/27 H7 N1 44 H7-3 A/FPV/Rostock/34 H7 N1 45 H7-4 A/FPV/Rostock/45/36 H7 N1 46 H7-5 A/chicken/Italy/444/99 H7 N1 47 H7-6 A/chicken/Italy/445/99 H7 N1 48 H7-7 A/chicken/Germany/R28/03 H7 N7 49 H7-8 A/duck/Alberta/48/76 H7 N3 50 H7-9 A/duck/Potsdam/13/80 H7 N7 51 H7-10 A/duck/Potsdam/15/80 H7 N7 52 H7-11 A/duck/Potsdam/62/81 H7 N7 53 H7-12 A/duck/Potsdam/64/81 H7 N3?N7? 54 H7-13 A/turkey/Ireland/PV8/98 H7 N7 55 H7-14 A/turkey/Italy/472/99 H7 N1 56 H7-15 A/chicken/Italy/473/99 H7 N1 57 H7-16 A/turkey/Germany/R11/01 H7 N7 58 H7-17 A/turkey/Italy/2043/03 H7 N3 59 H7-18 A/mallard/Germany/Wv K/03 H7 N3?N7? 60 H7-19 A/mallard/Germany/Wv38-41K/04 H7 N1 61 H7-20 A/equine/Prague/1/56 H7 N7 62 H8-1 A/turkey/Ontario/6118/68 H8 N4 63 H9-1 A/turkey/Wisconsin/66 H9 N2 64 H9-2 A/duck/Germany/113/95 H9 N2 65 H9-3 A/turkey/Germany/169/95 H9 N2 66 H9-4 A/chicken/Germany/45/98 H9 N2 67 H9-5 A/Pakistan/519 H9 N2 68 H10-1 A/quail/Italy/1117/65 H10 N8 69 H10-2 A/guinea fowl/hungary/1/69 H10 N8 70 H10-3 A/chicken/Germany/N/49 H10 N7 71 H10-4 A/Moorhen/Germ/Wv /03 H10 N4 72 H10-5 A/Mallard/Germ/Wv K/03 H10 N7 73 H11-1 A/duck/England/56 H11 N6 74 H11-2 A/Mallard/Germ/Wv K/03 H11 N9 75 H12-1 A/duck/Alberta/60/76 H12 N5 76 H13-1 A/gull/Maryland/704/77 H13 N6 77 H13-2 A/pilot whale/maine/328/84 H13 N2 78 H13-3 A/Gull/Germ/Wv KR/03 H13 79 H16-1 A/BHG/Sweden/5/99 H16 N3?

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5 /"&6 Lfd-Nr Val-Nr Isolat HA-type NA-type Avian H5-PCR nach H5-new-PCR Spackman et al. (2002) TC drlast TC drlast 9 H5-1 A/tern/S.Africa/61 H5 N3 19, , H5-2 A/chicken/Scotland/59 H5 N1 23, , H5-3 A/duck/Potsdam/2216/84 H5 N6 21, , H5-4 A/duck/Potsdam/2243/84 H5 N6 22, H5-6 A/duck/Potsdam/619/85 H5 N2 22, , H5-7 A/duck/Potsdam/1701/85 H5 N2 25, , H5-8 A/duck/Potsdam/1402/86 H5 N2 28, , H5-9 A/duck/Potsdam/1403/86 H5 N2 30, , H5-10 A/chicken/Italy/8/98 H5 N2 19, >42-19 H5-11 A/chicken/Italy/22/98 H5 N9 30, , H5-12 A/Hongkong/156/97 H5 N1 18, , H5-13 A/chicken/Indonesia/R /03 H5 N1 25, , H5-14 A/mallard/Germany/Wv K/03 H5 N2 26, >42-23 H5-15 A/mallard/Germany/Wv K/03 H5 N2?N3?N9? 23, , H5-16 A/teal/Germany/Wv K/03 H5 N2 22, >42-25 H5-17 A/mallard/Germany/Wv474-77K/04 H5 N? 24, , H5-18 A/chicken/Vietnam/P41/05 H5 N1 19, , H5-19 A/chicken/Vietnam/P78/05 H5 N1 19, , H5-20 A/chicken/Vietnam/P22/05 H5 N1 21, , H5-21 A/duck/Vietnam/TG36-H2/05 H5 N1 20, , H5-22 A/duck/Vietnam/AG40-O2/05 H5 N1 20, , H5-23 A/duck/Vietnam/TG24-O1/05 H5 N1 20, , H5-24 A/chicken/GXLA/1204/05 H5 N1 21, , H5-25 A/chicken/Indonesia/R132/1/04 H5 N1 33, , H5-26 A/chicken/Indonesia/R132/2/04 H5 N1 30, , H5-27 A/chicken/Indonesia/R132/3/04 H5 N1 35, , /"&6 '(/ /'(!& '(/ # #1 /6'(! /# * )*, & /

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8 ""&6 "'(!&'*) ) &". 6 /". (&... ) E' ) 2E5 /" 25 Well Name dr Last Ct (dr) Final Call (dr) H ,091 No Ct - H ,97 23,1 + H ,086 24,35 + H ,949 19,82 + H ,598 18,67 + H ,195 21,77 + H ,600 22,17 + H ,273 22,4 + H ,357 23,22 + H ,088 23,4 + H ,793 23,04 + H ,836 22,49 + H ,201 23,34 + H ,336 19,07 + H ,963 21,45 + H ,708 No Ct - H7-2 25,393 No Ct - H ,734 31,32 + H ,586 29,23 + H ,579 22,01 + H ,877 24,57 + H ,935 19,13 + H ,938 20,7 + H ,344 20,31 + NTC 22,686 No Ct - -$ "!'(!# * F "&6 (& 99;.,9 #: ) &", 9 9 & 6 ) #98'#.&&& 9#. */ 06;& 3./+" 0 1.'(/2 & $3 "405 " 4 6./+0 +" 5 '(4 ( 5.*7/! ".*8/.9/+:45 45 $3" ' & &1"40!4 55: $45 D

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10 AIV: Charakterisierung mittels RT-PCR und Sequenzierung HA0-Spaltstelle Signalpeptid S S Transmembranregion NH2-- HA1 HA2 --COOH AIV, Charakterisierung: Subtypspezifische RT-PCR-Teste HA1 HA1 S S HA2 HA2 H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11... H15 hohe Spezifität, gute Sensitivität >130 Isolate von Geflügel, Wild-und Ziervögeln ergänzt HAH (Monitoring-Programme!) ermöglicht Sequenzierung / erste Charakterisierung (Verwandschaft) Lee, M.-S. Et al., J Virol Meth 97 (2001),

11 AIV, Charakterisierung: RT-PCR-Teste für H5 und H7 HA0-Spaltstelle Signalpeptid S S Transmembranregion NH2-- HA1 HA2 --COOH H5-Lee H5-JN H5HA1 (+S) H5HA2 H7-JN H7HA-1 (+S) H7HA-2 AIV, Charakterisierung: RT-PCR-Teste für H5 Herkunft Isolat H5-Ja/Nein H5-Lee H5HA-1 H5HA-2 Material Indonesien R60/ AF Vietnam R75/ R76/ R76/ R77/ R78/ R79/ R80/ China R81/ Indonesien R133/03 + (+) + neg Organe R134/03 + (+) neg 3/7 + 1/1 + nd Tupfer WV Länder R121/2/ neg nd Tupfer AF

12 AIV, Charakterisierung: Sequenzierung H5-Isolate OIE, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Chapter : LPAIV LPNAI HPNAI Nicht-H5,-H7; gering- pathogen für Hühner H5, H7; IVPI<1.2 und HA 0 -Spaltstelle wie für gering-pathogene Viren H5, H7 u.a.; IVPI>1.2 oder HA 0 -Spaltstelle typisch für HPAIV AIV, Charakterisierung: Sequenzierung H5-Isolate A/ch/Italy/08/98 A/ch/Italy/22/98 A/dk/Potsd/619/85 WV /03 WV R121/2/05 A/ch/Vietnam/P78/05 A/ch/Indonesia/R60/05 V P Q R R R K K R * G L F V P Q K E T.. R * G L F V P Q R E T.. R * G L F V P Q R E T.. R * G L F V P Q R E T.. R * G L F Q R E R R. R K R * G L F Q R E R R R K K R * G L F 1

13 AIV, Charakterisierung: Sequenzierung H5-Isolate Phylogenetic tree of avian influenza virus isolates from Indonesia (raw data from GCG, Wisconsin Package Version 10.2) Isolates from Indonesia A/chicken/Indonesia/R134/03 ( H5INDO03 ) A/chicken/Indonesia/R60/05 ( H5INDO05 ) Sequences from GenBank AY AY AY AY DQ AY AY AY AY A/ch/Indonesia/BL/2003 A/ch/Indonesia/5/2004 A/goose/Thailand/79/2004 A/Thailand/1(KAN-1)/2004 A/ch/Thailand/73/2004 A/tiger/Thailand/SPB-1 A/ch/HongKong/61.9/02 A/ch/Hubai/489/2004 A/ch/Guangdong/174/04 A/ch/Indonesia/R134/03 and A/ch/Indonesia/R69/05 show a homology of 98.58% on nucleotide level (1482 bp). The homology between the Indonesian sequences from GenBank and A/ch/Indonesia/R60/05 amounts and 98.56%, respectively. Compared to the isolates from China, the homology is about 97.5%, and with the sequences from Thailand it is about 96.7%. No differences are seen with regard to HA cleavage site, potential glycosilation sites and receptor binding sites between the own Indonesian sequences and those from GenBank. AIV, Charakterisierung: Sequenzierung H5-Isolate AIV, H5: Phylogenetischer Baum ausgewählter europäischer, asiatischer und amerikanischer Isolate (Rohdaten; GCG, Wisconsin Package Version 10.2) AF A/gull/Pennsylvenia/4175/83 H5N1, lp AF A/duck/Minnesota/1525/81 H5N1, lp X07826 A/chicken/Scotland/59 H5N1, hp AF A/duck/Potsdam/2216-4/84 H5N2, lp AF A/duck/Potsdam/1402-6/86 H5N2, lp S68489 A/turkey/England/50-92/91 H5N1, hp AJ A/chicken/Italy/8/98 H5N2, hp AY A/goose/Thailand/79/2004 H5N1, hp DQ A/chicken/Thailand/73/2004 H5N1, hp AY A/chicken/Indonesia/BL72003 H5N1, hp AY A/chicken/Indonesia/5/2004 H5N1, hp AY A/chicken/Guangdong/174/04 H5N1, hp DQ A/goose/Novosibirsk/4/2005 H5N1, hp AY77079 A/chicken/Hubai/489/2004 H5N1, hp?

14 AIV: Charakterisierung mittels RT-PCR und Sequenzierung H5- und H7-PCR wenn H5- oder H7-positiv: Geflügelpestverdacht (bei Sekundärausbrüchen Bestätigung GP) direkte Sequenzierung LPAIV oder HPAIV ggf. Bestätigung Geflügelpest HA1 HA2 B