V IROLOG ICAS IN ICA2014 DO I10 .1007 /s12250 -014 -3444 -8
LETTER S imu l taneousde tec t iono fduckhepa t i t isAv i rustypes1and3 , ando fduckas t rov i rustype1 ,by mu l t ip lexRT -PCR D ea rEd i to r , Du ckv i ru sh ep a t i t i s(DVH )i sc au s edbya tl e a s tth r e e d i f f e r en tRNAv i ru s e s ,in c lud ingdu ckh ep a t i t i sAv i ru s (DHAV ) ,du cka s t rov i ru styp e1(DA s tV -1 ) ,anddu ck a s t rov i ru styp e2(DA s tV -2 ) .Th ef i r s to fth e s e , DHAV , h a sb e enc l a s s ifiedin toth r e es e ro typ e sbyn eu t r a l i z a t ion t e s t s :typ e1(DHAV -1 ) ,typ e2(DHAV -2 ) ,andtyp e3 (DHAV -3 )(W angL ,e ta l . ,2008 ) .Twoo fth e s es e ro typ e s , DHAV -1and DHAV -3 ,tog e th e rw i th DA s tV -1 , h av eb e eni so l a t edf romv a r iou sd i f f e r en tr eg ion so f Ch in ainr e c en ty e a r s(Ch enL ,e ta l . ,2012 ;Ch enLL ,e t a l . ,2013 ;D ingC ,e ta l . ,2007 ;FuY ,e ta l . ,2008 ,2009 ; G aoJ ,e ta l . ,2012 ;W angL ,e ta l . ,2008 ;W angX ,e ta l . , 201 1 ;XuQ ,e ta l . ,2012 ) . Indu ck l ing s ,i ti sd i f f i cu l tu s ingconv en t ion a ld i ag no s t i cm e thod stod e t e rm in e wh i chp a r t i cu l a rp a thog en i sr e spon s ib l efo ranin c id en c eo f DVH ,b e c au s eth e c l in i c a lind i c a t ion sandg ro s sp a tho log i c a lch ang e sa r e s im i l a rfo ra l lth r e etyp e so fdu ckv i ru sh ep a t i t i s(DHV ) in f e c t ion s .Anumb e ro fl abo r a to ry m e thod sfo rth e mo l e cu l a rd e t e c t iono fDHVin f e c t ionh av eb e end ev e lop ed r e c en t lyanda r enowincommonu s e(Ch engAC ,e ta l . , 2009 ;Ch enLL ,e ta l . ,2013 ;Hu angQ ,e ta l . ,2012 ;K im MC ,e ta l . ,2007 ,2008 ;Y ang M ,e ta l . ,2008 ) ;how ev e r , th eya l lsh a r ea m a jo rd i s adv an t ag einth a tth e i rab i l i ty tod i s t ingu i shb e tw e en DHAV -1 , DHAV -3 ,and DA s tV -1 inas ing l er e a c t ioni sl im i t ed .Inth i ss tudy ,th e r e fo r e ,a m u l t i p l e xr e v e r s et r a n s c r i p t i o np o l ym e r a s ec h a i nr e a c t i o n (RT -PCR )a s s ayh a sb e end ev e lop edth a tc and e t e c tand d i s t ingu i sh DHAV -1 , DHAV -3and DA s tV -1s imu l t an e ou s ly . Th r e ep a i r so fp r im e r sfo r DHAV -1 , DHAV -3 ,and DA s tV -1 w e r es e l e c t edonth eb a s i so f who l e -g enom e s equ en c ea l ignm en t so f DHVs equ en c e sr e t r i ev edf rom G enB ank(h t tp : / /www .n cb i .n lm .n ih .gov ) .T oid en t i fy ands e l e c tsu i t ab l ep r im e r s ,57 DHAV -1s equ en c e s ,20 DHAV -3s equ en c e s ,and6 DA s tV -1s equ en c e sw e r e a l ign ed .Th ep r im e rp a i r sfo rth eth r e ev i ru s e s[DHAV 1F :5 ’ -GATGTGGCAY (T /C )GTTGTY (T /C )AAY (T / C )CGA -3 ’ ; DHAV -1R :5 ’ -CTGATGTD (G /A /T )CCAG GR (A /G )A TTGGTCG 3 ’ ;DHAV 3F :5 ’ -GAGCCAGAA TTGGAATGGACACA -3 ’ ; DHAV -3R :5 ’ -CATACT TR (G /A )CCACCAACTGCCAATC -3 ’ ; DA s tV -1F : ©W IV ,CASandSp r inge r -Ve r lagBe r l inHe ide lbe rg2014
5 ’ -ATGGCCCAGAGCGGTGAAAA -3 ’ ;and DA s tV 1R :5 ’ -GCCAGGTGTCAACAATCATGC -3 ’ ]w e r ed e s ign edtoamp l i fy570bp ,1099bpand898bpf r agm en t s , r e sp e c t iv e ly .A l lo fth ep r im e r sw e r esyn th e s i z edby S angon(Sh angh a i ,Ch in a ) . DHAV -1s t r a inLY0801( a c c e s s ionno .F J436047 ) , DHAV -3s t r a inSD1 101( a c c e s s ionno .JQ409566 ) ,and DA s tV -1s t r a in WF1201( a c c e s s ionno .JX439643 )w e r e s e l e c t eda sr e f e r en c ei so l a t e s(Ch enLL ,e ta l . ,2013 ;Xu Q ,e ta l . ,2012 ) .Th eth r e ev i ru s e sw e r ep rop ag a t edin h e a l thy1 -d ay -o lddu ck l ing sbyin t r amu s cu l a rin j e c t ion . A t24hpo s t ino cu l a t ion ,th edu ck l ing sb eg antod i eand th e i rl iv e r sw e r eco l l e c t ed .RNAw a sth enex t r a c t edu s inganEZNAV i r a lRNAK i t(Om eg aB io -T ek ,Do r av i l l e , GA , USA ) ,a c co rd ingtoth em anu f a c tu r e r ’ sin s t ru c t ion s .R ev e r s et r an s c r ip t ion(RT )w a sc a r r i edou tu s inga R ev e r tA idF i r s tS t r andcDNASyn th e s i sK i t(F e rm en t a s / Th e rmoS c i en t i f i c ,W a l th am , MA , USA ) ,a c co rd ingto th em anu f a c tu r e r ’ sd i r e c t ion s .Th er e a c t ion m ix tu r econ t a in ed8μLo fRNAand1μMDHAV -1F ,DHAV -3Fand DA s tV -1Fp r im e r s ,inafin a lvo lum eo f20μL .Th eRT s t e pw a sc a r r i e do u ta t4 2 °Cf o r6 0m i na n dw a sf o l l ow e d bya5 m inp e r ioda t70 °C .F in a l ly ,a mu l t ip l exPCRa s s ay w a sc a r r i edou tinavo lum eo f50μLinar e a c t ion m ix tu r ecomp r i s ing50 mmo l /L KC l ,10 mmo l /LT r i s 0 0μm o l /LdNTP s ,0 . 4 HC l( pH8 . 3 ) ,2 . 5mm o l /L M gC l2,2 μmo l /L DHAV -1F /R ,2 .5μmo l /L DHAV -3F /R ,3μmo l / L DA s tV -1F /R ,2 .0 Uo fT aq DNApo lym e r a s e ,and3 μLo fcDNA .Th eev en tu a lop t im i z edcy c l ingcond i t ion s w e r e :95 °Cfo r5 m in ;fo l low edby32cy c l e sa t95 °Cfo r 30s ,62 °Cfo r30s ,and72 °Cfo r1 m in ;andfin a l ly72 °C fo r10 m in .Th eamp l ifi edp rodu c t sw e r ee l e c t ropho r e s ed ona0 .8%ag a ro s eg e landv i su a l i z edw i the th id iumb ro m id e . N ex t ,th ed e t e c t ions en s i t iv i tyo fth e mu l t ip l ex RT PCRp ro c edu r ew a sd e t e rm in ed ,u s ingbo thto t a lt i s su e RNAandv i r a lg enom eequ iv a l en t s .Inth ec a s eo fto t a l t i s su e RNA ,th ePCRexp e r im en tw a sp e r fo rm ed w i th s e r i a l10 fo ldd i lu t ion s(1μg–1pg )o fto t a lRNAex t r a c t edf roml iv e rs amp l e sth a th adb e ena r t ific i a l lyin f e c t ed w i th DHAV -1 , DHAV -3 ,and DA s tV -1 .Th ed e t e c t ion s en s i t iv i ty w a sob s e rv edtob e10pgo fto t a lRNAfo r F igu r e1A) .Inth ec a s e e a cho fth eth r e etyp e so fv i ru s e s( o fv i r a lg enom eequ iv a l en t s ,cRNAs t and a rd sfo rDHAV 1
Mu l t ip lexRT -PCRfo rduckhepa t i t isv i ruses
F igu re1 . Sens i t iv i tyandspec ific i tyo fthe mu l t ip lex RT -PCR .A : Sens i t iv i tyo fthe mu l t ip lex RT -PCRus ingto ta l l ive rt issue RNAasatemp la te .M , DNA ma rke r ;1 ,1μg ;2 ,100ng ;3 ,10ng ;4 ,1ng ;5 ,100pg ;6 ,10pg ;and7 , 10 1 1pg .B :Sens i t iv i tyo fthe mu l t ip lex RT -PCRp repa redf rom10 –10 cop ieso fcRNAfo reacho f DHAV -1 , DHAV 10 9 8 7 3 ,and DAs tV -1usedasas ta r t ingtemp la te .M , DNA ma rke r ;1 ,10 cop ies ;2 ,10 cop ies ;3 ,10 cop ies ;4 ,10 6 5 4 3 2 1 cop ies ;5 ,10 cop ies ;6 ,10 cop ies ;7 ,10 cop ies ;8 ,10 cop ies ;9 ,10 cop ies ;and10 ,10 cop ies .C : Mu l t ip lex PCR -amp l ifiedp roduc tsf rompu r ified DNAs /RNAso fknownduckpa thogens .M , DL2000 DNA ma rke r ;1 , DHAV 1 , DHAV -3 ,and DAs tV -1 ;2 , DHAV -1and DHAV -3 ;3 , DHAV -1and DAs tV -1 ;4 , DHAV -3and DAs tV -1 ;5 , DHAV -1 ; 6 , DHAV -3 ;7 , DAs tV -1 ;8 ,nega t ivecon t ro l(hea l thyduck l ingl ive rsamp le ) ;9 ,av ianinfluenzav i rus ;10 , Newcas t le d iseasev i rus ;1 1 ,duckp laguev i rus ;12 ,duckc i rcov i rus ;13 ,R ieme re l laana t ipes t i fe r;and14 ,Esche r ich iaco l i.
1 ,DHAV -3 ,andDA s tV -1w e r econ s t ru c t ed ,s t a r t ingw i th th r e ep a i r so fp r im e r sd e s ign edfo rth i spu rpo s e[DHAV 1comFSP65 ’ -CCCAAGCTTATACGATTTAGGTGA CACTATAGGATGTGGCAY (T /C )GTTGTY (T /C ) AAY (T /C )CGA -3 ’ ; DHAV -1comR5 ’ -CGGGATCCC TGATGTD (G /A /T )CCAGGR (A /G )ATTGGTCG -3 ’ ; DHAV -3comFSP65 ’ -CCCAAGCTTATACGATTTAGG TGACACTATAGGAGCCAGAATTGGAATGGACAC A -3 ’ ; DHAV -3comR5 ’ -CGGGATCCCATACTTR (G / A )CCACCAACTGCCAATC -3 ’ ; DA s tV -1comFSP6 5 ’ -CCCAAGCTTATACGATTTAGGTGACACTATAGA TGGCCCAGAGCGGTGAAAA -3 ’ ;andDA s tV -1comR 5 ’ -CGGGATCCGGAGCATGCACAAGTGTTCATC A -3 ’ ;th eSP6p romo t e rs equ en c ei sind i c a t edini t a l i c s ] .Th einv i t r ot r an s c r ip t iono fth eRNA sfo r DHAV 2
1 , DHAV -3 ,and DA s tV -1 w a sc a r r i edou tu s ingth e R iboMAXL a rg es c a l e RNAP rodu c t ionSy s t em -SP6 andT7k i t(P rom eg a ,M ad i son ,W I , USA ) ,a c co rd ingto th em anu f a c tu r e r ’ sin s t ru c t ion s .U s ing10 fo lds e r i a ld i 10 1 –10 cop i e s )o fth esyn th e t i ccRNA m ix tu r e lu t ion s(10 a st emp l a t e ,th elow e s td e t e c t ionl im i to fth e mu l t ip l ex i e sfo re a cho fth e RT -PCRw a sd e t e rm in edtob e102 cop th r e etyp e so fv i ru s e s( F igu r e1B) . Th esp e c i f i c i tyo fth e mu l t ip l exRT -PCR w a sa s c e r t a in edbyt e s t ingi tag a in s tar ang eo fo th e rdu ckp a tho g en sth a tc anb ep r e s en tdu r ing mu l t ip l ein f e c t ion sw i th DHAV -1 ,DHAV -3 ,andDA s tV -1 ,in c lud ingdu ckp l agu e v i ru s ,av i anin f lu en z av i ru s(H9N2 ) ,N ew c a s t l ed i s e a s e v i ru s ,th ecomp l e t eg enom i c DNAo fdu ckc i r cov i ru s s t r a inF J0601 ,R i em e r e l laana t ip e s t i f e r andE s ch e r i ch ia V IROLOG ICAS IN ICA
L in l inChene ta l
co l i .A l lo fth e s eo th e rp a thog ens amp l e st e s t edn eg a t iv e , ex c ep tinth ec a s eo fadm ix tu r ew i thon e(o r mo r e )o f DHAV -1 ,DHAV -3 ,andDA s tV -1(F igu r e1C) . Ato t a lo f48c l in i c a ll iv e rs amp l e sw i thsu sp e c t ed v i r a lh ep a t i t i sin f e c t ionw e r eth ent e s t edino rd e rtoev a l u a t eth ep e r fo rm an c eo fth e mu l t ip l exRT -PCRa s s ay .In add i t ion ,as imp l exRT -PCRa s s ay ,v i r a lcu l tu r e ,anda n eu t r a l i z a t iont e s tw e r ee a chund e r t ak enonth e48s am p l e s ,a sp r ev iou s lyd e s c r ib ed(Ch enLL ,e ta l . ,2013 ) . Th ep r im e r su s edinth es imp l ex RT -PCRa s s ay sw e r e th es am ea sfo rth e mu l t ip l ex RT -PCRa s s ay . Ov e r a l l , e igh ts amp l e sw e r eid en t ifieda sco in f e c t edw i thDHAV 1 /DHAV -3 ,1s amp l ew a sid en t ifi eda sco in f e c t ed w i th DHAV -1 /DA s tV -1 ,3s amp l e sw e r eid en t i f i eda sco in f e c t ed w i th DHAV -3 /DA s tV -1 ,and36s amp l e sw e r e showntob epo s i t iv efo re i th e r DHAV -1(10s amp l e s ) , DHAV -3(18s amp l e s ) ,o r DA s tV -1(8s amp l e s )a lon e . Nos amp l ew a sfoundtob eco in f e c t edw i tha l lth eth r e e v i ru s e stog e th e r .Th er e su l t sw e r econfi rm edbys imp l ex RT -PCR ,v i r a li so l a t ion ,andan eu t r a l i z a t iont e s t–and w i th100%ag r e em en t . DVHi so fe conom i cimpo r t an c eina l ldu ck r e a r ing r eg ion so fth e wo r ld .InCh in a ,in f e c t ionh a sb e ena s so c i a t ed w i thth r e etyp e so fv i ru s : DHAV -1 , DHAV -3 ,and DA s tV -1(Ch enL ,e ta l . ,2012 ;Ch enLL ,e ta l . ,2013 ; D ingC ,e ta l . ,2007 ;FuY ,e ta l . ,2008 ,2009 ;G aoJ ,e t a l . ,2012 ;W angL ,e ta l . ,2008 ;W angX ,e ta l . ,201 1 ;Xu Q ,e ta l . ,2012 ) . Du ck l ing sin f e c t edbyanyon eo fth e s e th r e ev i ru s e sc anexh ib i ts im i l a rsymp tom sind iv idu a l ly , su cha sasho r tin cub a t ionp e r iod ,sudd enon s e t ,h igh mo r t a l i ty ,op i s tho tonu s ,anden l a r g edl iv e rw i thd i s t in c t e c chymo t i ch emo r rh ag e s . Ap r e sump t iv ed i agno s i so f DVHc anb em ad eb a s edonth eh i s to ryandl e s ion s ,bu t i ti sd i f f i cu l ttoa s c e r t a in wh i chtyp e(o rtyp e s )o fv i ru si sc au s ingth ein f e c t ion .In2008 ,a mu l t ip l exPCR w a sd ev e lop edtod i f f e r en t i a t eb e tw e enth e DHAV -1 and DHAV -3s t r a in s(K im MC ,e ta l . ,2008 ) ;and mo r e r e c en t ly ,w ed ev e lop edanimp rov eddup l ex RT -PCR m e thodth a tf a c i l i t a t e sth er ap idandco s t e f f e c t iv el abo r a to ryd e t e c t iono fm ix edin f e c t ion sc au s edbyd i f f e r en t DHAV -1andDHAV -3s t r a in sindu ck l ing s(Ch enLL ,e t a l . ,2013 ) .N e i th e ro fth e s e mu l t ip l exPCRa s s ay scou ld d e t e c tDA s tV -1 ,how ev e r . Inth ep r e s en ts tudy ,a mu l t ip l ex RT -PCRh a sb e en d ev e lop edfo rth es imu l t an eou sd e t e c t iono f DHAV 1 , DHAV -3 ,and DA s tV -1inc l in i c a lsp e c im en s .T oth e b e s to fou rknow l edg e ,th i si sth ef i r s td e s c r ip t iono f a mu l t ip l ex RT -PCRth a tc and e t e c tth eth r e etyp e so f DVHv i ru s e s .Th e mu l t ip l ex RT -PCR m e thodi s DVH v i ru s sp e c i f i canddo e sno tamp l i fyg enom i c DNAo r RNAf romo th e rdu ckp a thog en s .I t sd e t e c t ionl im i ti s 2 e s t im a t edtob e10pgo fto t a ll iv e rt i s su eRNA ,o r10 cop i e so fe a cho f DHAV -1 , DHAV -3and DA s tV -1v i r a l RNA .Fu r th e rmo r e ,ad i f f e r en t i a ld i agno s i so f DHAV www .v i ros in .o rg
1 , DHAV -3 ,and DA s tV -1 m ayb eob t a in edinas ing l e r e a c t ion w i th inaf ewhou r s , wh e r e a sd i f f e r en t i a t iono f th eth r e ev i ru s e sfo l low edbyc ro s s -n eu t r a l i z a t iont e s t s t ak e ss ev e r a ld ay sa tl e a s t .Th i s mu l t ip l exPCR m e thod i sth e r e fo r ear ap id ,e f f e c t iv eandp r a c t i c a ltoo lfo rth e d i f f e r en t i a ld i agno s i so fm ix edin f e c t ion sw i thth eth r e e typ e so f DVHv i ru s e s ,andfo rth eep id em io log i c a lsu r v e i l l an c eo fDVH . FOOTNOOTES Th i ss tudy w a sfund edbyg r an t sf romth eSh andong Mod e rn Ag r i cu l tu r a lT e chno logy &Indu s t rySy s t em(SDA IT -13 -01 1 -15 ) andth eS c i en c eandT e chno logy Comm i s s iono fSh andong P rov in c e(2010GNC10914 ) ,Ch in a . A l lth eau tho r sd e c l a r eth a tth eyh av enocomp e t ingin t e r e s t .A l l in s t i tu t ion a landn a t ion a lgu id e l in e sfo rth ec a r eandu s eo fl abo r a to ryan im a l sw e r efo l low ed . 1 ,2# 1 ,2 L in l in Ch en1,2#, M ing j i eM a , Ru ihu aZh ang ,Q i an 1 ,2 3 4 1 ,2 Xu , X ingku iS i, Y uW ang, Zh i j ing X i e ,Sh i j in 1 ,2 J i ang
1 .D ep a r tm en to fP r ev en t iv eV e t e r in a ry M ed i c in e , Co l l eg eo f V e t e r in a ry M ed i c in e ,Sh andong Ag r i cu l tu r a l Un iv e r s i ty ,T a i an 271018 ,Ch in a 2 .Sh andongP rov in c i a lK eyL abo r a to ryo fAn im a lB io t e chno logy andD i s e a s eCon t ro landP r ev en t ion ,T a i an271018 ,Ch in a 3 . Depa r tmen to f An ima l Med ic ine , Fo shan Sc ienceand T e chno logyUn iv e r s i ty ,Fo sh an528231 ,Ch in a 4 .D ep a r tm en to fB a s i cM ed i c a lS c i en c e s ,T a i sh an M ed i c a l Co l l eg e ,T a i an271000 ,Ch in a Co r r e spond en c e : Phon e :+86 -538 -8245799 ;F ax :+86 -538 -8245799 . Em a i l :s j j i ang@ sd au . edu . cn # Th e s eau tho r scon t r ibu t edequ a l lytoth i swo rk . Pub l i sh edon l in e :22 M ay2014
REFERENCES Ch enL ,XuQ ,Zh angR ,e ta l .2012 .JV i ro l ,86 :13833 -13834 . Ch enLL ,XuQ ,Zh angRH ,e ta l .2013 .JV i ro lM e thod s ,192 :12 -17 . Ch engAC ,W ang MS ,X in H Y ,e ta l .2009 .J M i c rob io lM e thod s ,77 : 332 -336 . D ingC ,Zh angD .2007 .V i ro logy ,361 :9 -17 . FuY ,P an M ,W angX ,e ta l .2008 .V e tM i c rob io l ,131 :247 -257 . FuY ,P an M ,W angX ,e ta l .2009 .JG enV i ro l ,90 ,1 104 -1 108 . G aoJ ,Ch enJ ,S iX ,e ta l .2012 .V i ro lS in ,27 :248 -253 . Hu angQ ,Y u eH ,Zh angB ,e ta l .2012 .50 :3318 -3323 . K im MC ,KwonYK ,JohSJ ,e ta l .2007 .A v i anD i s ,51 :540 -545 . K im MC ,KwonYK ,JohSJ ,e ta l .2008 .A v i anP a tho l ,37 :171 -177 . W angL ,P an M ,FuY ,e ta l .2008 .V i ru sG en e s ,37 :52 -59 . W angX ,W angY ,X i eX ,e ta l .201 1 .JV i ro lM e thod s ,171 :8 -12 XuQ ,Zh angR ,Ch enL ,e ta l .2012 .JV i ro l ,86 :13848 . Y ang M ,Ch engA ,W ang M ,e ta l .2008 .JV i ro lM e thod s ,153 :55 -60 .
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LETTER S imu l taneousde tec t iono fduckhepa t i t isAv i rustypes1and3 , ando fduckas t rov i rustype1 ,by mu l t ip lexRT -PCR D ea rEd i to r , Du ckv i ru sh ep a t i t i s(DVH )i sc au s edbya tl e a s tth r e e d i f f e r en tRNAv i ru s e s ,in c lud ingdu ckh ep a t i t i sAv i ru s (DHAV ) ,du cka s t rov i ru styp e1(DA s tV -1 ) ,anddu ck a s t rov i ru styp e2(DA s tV -2 ) .Th ef i r s to fth e s e , DHAV , h a sb e enc l a s s ifiedin toth r e es e ro typ e sbyn eu t r a l i z a t ion t e s t s :typ e1(DHAV -1 ) ,typ e2(DHAV -2 ) ,andtyp e3 (DHAV -3 )(W angL ,e ta l . ,2008 ) .Twoo fth e s es e ro typ e s , DHAV -1and DHAV -3 ,tog e th e rw i th DA s tV -1 , h av eb e eni so l a t edf romv a r iou sd i f f e r en tr eg ion so f Ch in ainr e c en ty e a r s(Ch enL ,e ta l . ,2012 ;Ch enLL ,e t a l . ,2013 ;D ingC ,e ta l . ,2007 ;FuY ,e ta l . ,2008 ,2009 ; G aoJ ,e ta l . ,2012 ;W angL ,e ta l . ,2008 ;W angX ,e ta l . , 201 1 ;XuQ ,e ta l . ,2012 ) . Indu ck l ing s ,i ti sd i f f i cu l tu s ingconv en t ion a ld i ag no s t i cm e thod stod e t e rm in e wh i chp a r t i cu l a rp a thog en i sr e spon s ib l efo ranin c id en c eo f DVH ,b e c au s eth e c l in i c a lind i c a t ion sandg ro s sp a tho log i c a lch ang e sa r e s im i l a rfo ra l lth r e etyp e so fdu ckv i ru sh ep a t i t i s(DHV ) in f e c t ion s .Anumb e ro fl abo r a to ry m e thod sfo rth e mo l e cu l a rd e t e c t iono fDHVin f e c t ionh av eb e end ev e lop ed r e c en t lyanda r enowincommonu s e(Ch engAC ,e ta l . , 2009 ;Ch enLL ,e ta l . ,2013 ;Hu angQ ,e ta l . ,2012 ;K im MC ,e ta l . ,2007 ,2008 ;Y ang M ,e ta l . ,2008 ) ;how ev e r , th eya l lsh a r ea m a jo rd i s adv an t ag einth a tth e i rab i l i ty tod i s t ingu i shb e tw e en DHAV -1 , DHAV -3 ,and DA s tV -1 inas ing l er e a c t ioni sl im i t ed .Inth i ss tudy ,th e r e fo r e ,a m u l t i p l e xr e v e r s et r a n s c r i p t i o np o l ym e r a s ec h a i nr e a c t i o n (RT -PCR )a s s ayh a sb e end ev e lop edth a tc and e t e c tand d i s t ingu i sh DHAV -1 , DHAV -3and DA s tV -1s imu l t an e ou s ly . Th r e ep a i r so fp r im e r sfo r DHAV -1 , DHAV -3 ,and DA s tV -1 w e r es e l e c t edonth eb a s i so f who l e -g enom e s equ en c ea l ignm en t so f DHVs equ en c e sr e t r i ev edf rom G enB ank(h t tp : / /www .n cb i .n lm .n ih .gov ) .T oid en t i fy ands e l e c tsu i t ab l ep r im e r s ,57 DHAV -1s equ en c e s ,20 DHAV -3s equ en c e s ,and6 DA s tV -1s equ en c e sw e r e a l ign ed .Th ep r im e rp a i r sfo rth eth r e ev i ru s e s[DHAV 1F :5 ’ -GATGTGGCAY (T /C )GTTGTY (T /C )AAY (T / C )CGA -3 ’ ; DHAV -1R :5 ’ -CTGATGTD (G /A /T )CCAG GR (A /G )A TTGGTCG 3 ’ ;DHAV 3F :5 ’ -GAGCCAGAA TTGGAATGGACACA -3 ’ ; DHAV -3R :5 ’ -CATACT TR (G /A )CCACCAACTGCCAATC -3 ’ ; DA s tV -1F : ©W IV ,CASandSp r inge r -Ve r lagBe r l inHe ide lbe rg2014
5 ’ -ATGGCCCAGAGCGGTGAAAA -3 ’ ;and DA s tV 1R :5 ’ -GCCAGGTGTCAACAATCATGC -3 ’ ]w e r ed e s ign edtoamp l i fy570bp ,1099bpand898bpf r agm en t s , r e sp e c t iv e ly .A l lo fth ep r im e r sw e r esyn th e s i z edby S angon(Sh angh a i ,Ch in a ) . DHAV -1s t r a inLY0801( a c c e s s ionno .F J436047 ) , DHAV -3s t r a inSD1 101( a c c e s s ionno .JQ409566 ) ,and DA s tV -1s t r a in WF1201( a c c e s s ionno .JX439643 )w e r e s e l e c t eda sr e f e r en c ei so l a t e s(Ch enLL ,e ta l . ,2013 ;Xu Q ,e ta l . ,2012 ) .Th eth r e ev i ru s e sw e r ep rop ag a t edin h e a l thy1 -d ay -o lddu ck l ing sbyin t r amu s cu l a rin j e c t ion . A t24hpo s t ino cu l a t ion ,th edu ck l ing sb eg antod i eand th e i rl iv e r sw e r eco l l e c t ed .RNAw a sth enex t r a c t edu s inganEZNAV i r a lRNAK i t(Om eg aB io -T ek ,Do r av i l l e , GA , USA ) ,a c co rd ingtoth em anu f a c tu r e r ’ sin s t ru c t ion s .R ev e r s et r an s c r ip t ion(RT )w a sc a r r i edou tu s inga R ev e r tA idF i r s tS t r andcDNASyn th e s i sK i t(F e rm en t a s / Th e rmoS c i en t i f i c ,W a l th am , MA , USA ) ,a c co rd ingto th em anu f a c tu r e r ’ sd i r e c t ion s .Th er e a c t ion m ix tu r econ t a in ed8μLo fRNAand1μMDHAV -1F ,DHAV -3Fand DA s tV -1Fp r im e r s ,inafin a lvo lum eo f20μL .Th eRT s t e pw a sc a r r i e do u ta t4 2 °Cf o r6 0m i na n dw a sf o l l ow e d bya5 m inp e r ioda t70 °C .F in a l ly ,a mu l t ip l exPCRa s s ay w a sc a r r i edou tinavo lum eo f50μLinar e a c t ion m ix tu r ecomp r i s ing50 mmo l /L KC l ,10 mmo l /LT r i s 0 0μm o l /LdNTP s ,0 . 4 HC l( pH8 . 3 ) ,2 . 5mm o l /L M gC l2,2 μmo l /L DHAV -1F /R ,2 .5μmo l /L DHAV -3F /R ,3μmo l / L DA s tV -1F /R ,2 .0 Uo fT aq DNApo lym e r a s e ,and3 μLo fcDNA .Th eev en tu a lop t im i z edcy c l ingcond i t ion s w e r e :95 °Cfo r5 m in ;fo l low edby32cy c l e sa t95 °Cfo r 30s ,62 °Cfo r30s ,and72 °Cfo r1 m in ;andfin a l ly72 °C fo r10 m in .Th eamp l ifi edp rodu c t sw e r ee l e c t ropho r e s ed ona0 .8%ag a ro s eg e landv i su a l i z edw i the th id iumb ro m id e . N ex t ,th ed e t e c t ions en s i t iv i tyo fth e mu l t ip l ex RT PCRp ro c edu r ew a sd e t e rm in ed ,u s ingbo thto t a lt i s su e RNAandv i r a lg enom eequ iv a l en t s .Inth ec a s eo fto t a l t i s su e RNA ,th ePCRexp e r im en tw a sp e r fo rm ed w i th s e r i a l10 fo ldd i lu t ion s(1μg–1pg )o fto t a lRNAex t r a c t edf roml iv e rs amp l e sth a th adb e ena r t ific i a l lyin f e c t ed w i th DHAV -1 , DHAV -3 ,and DA s tV -1 .Th ed e t e c t ion s en s i t iv i ty w a sob s e rv edtob e10pgo fto t a lRNAfo r F igu r e1A) .Inth ec a s e e a cho fth eth r e etyp e so fv i ru s e s( o fv i r a lg enom eequ iv a l en t s ,cRNAs t and a rd sfo rDHAV 1
Mu l t ip lexRT -PCRfo rduckhepa t i t isv i ruses
F igu re1 . Sens i t iv i tyandspec ific i tyo fthe mu l t ip lex RT -PCR .A : Sens i t iv i tyo fthe mu l t ip lex RT -PCRus ingto ta l l ive rt issue RNAasatemp la te .M , DNA ma rke r ;1 ,1μg ;2 ,100ng ;3 ,10ng ;4 ,1ng ;5 ,100pg ;6 ,10pg ;and7 , 10 1 1pg .B :Sens i t iv i tyo fthe mu l t ip lex RT -PCRp repa redf rom10 –10 cop ieso fcRNAfo reacho f DHAV -1 , DHAV 10 9 8 7 3 ,and DAs tV -1usedasas ta r t ingtemp la te .M , DNA ma rke r ;1 ,10 cop ies ;2 ,10 cop ies ;3 ,10 cop ies ;4 ,10 6 5 4 3 2 1 cop ies ;5 ,10 cop ies ;6 ,10 cop ies ;7 ,10 cop ies ;8 ,10 cop ies ;9 ,10 cop ies ;and10 ,10 cop ies .C : Mu l t ip lex PCR -amp l ifiedp roduc tsf rompu r ified DNAs /RNAso fknownduckpa thogens .M , DL2000 DNA ma rke r ;1 , DHAV 1 , DHAV -3 ,and DAs tV -1 ;2 , DHAV -1and DHAV -3 ;3 , DHAV -1and DAs tV -1 ;4 , DHAV -3and DAs tV -1 ;5 , DHAV -1 ; 6 , DHAV -3 ;7 , DAs tV -1 ;8 ,nega t ivecon t ro l(hea l thyduck l ingl ive rsamp le ) ;9 ,av ianinfluenzav i rus ;10 , Newcas t le d iseasev i rus ;1 1 ,duckp laguev i rus ;12 ,duckc i rcov i rus ;13 ,R ieme re l laana t ipes t i fe r;and14 ,Esche r ich iaco l i.
1 ,DHAV -3 ,andDA s tV -1w e r econ s t ru c t ed ,s t a r t ingw i th th r e ep a i r so fp r im e r sd e s ign edfo rth i spu rpo s e[DHAV 1comFSP65 ’ -CCCAAGCTTATACGATTTAGGTGA CACTATAGGATGTGGCAY (T /C )GTTGTY (T /C ) AAY (T /C )CGA -3 ’ ; DHAV -1comR5 ’ -CGGGATCCC TGATGTD (G /A /T )CCAGGR (A /G )ATTGGTCG -3 ’ ; DHAV -3comFSP65 ’ -CCCAAGCTTATACGATTTAGG TGACACTATAGGAGCCAGAATTGGAATGGACAC A -3 ’ ; DHAV -3comR5 ’ -CGGGATCCCATACTTR (G / A )CCACCAACTGCCAATC -3 ’ ; DA s tV -1comFSP6 5 ’ -CCCAAGCTTATACGATTTAGGTGACACTATAGA TGGCCCAGAGCGGTGAAAA -3 ’ ;andDA s tV -1comR 5 ’ -CGGGATCCGGAGCATGCACAAGTGTTCATC A -3 ’ ;th eSP6p romo t e rs equ en c ei sind i c a t edini t a l i c s ] .Th einv i t r ot r an s c r ip t iono fth eRNA sfo r DHAV 2
1 , DHAV -3 ,and DA s tV -1 w a sc a r r i edou tu s ingth e R iboMAXL a rg es c a l e RNAP rodu c t ionSy s t em -SP6 andT7k i t(P rom eg a ,M ad i son ,W I , USA ) ,a c co rd ingto th em anu f a c tu r e r ’ sin s t ru c t ion s .U s ing10 fo lds e r i a ld i 10 1 –10 cop i e s )o fth esyn th e t i ccRNA m ix tu r e lu t ion s(10 a st emp l a t e ,th elow e s td e t e c t ionl im i to fth e mu l t ip l ex i e sfo re a cho fth e RT -PCRw a sd e t e rm in edtob e102 cop th r e etyp e so fv i ru s e s( F igu r e1B) . Th esp e c i f i c i tyo fth e mu l t ip l exRT -PCR w a sa s c e r t a in edbyt e s t ingi tag a in s tar ang eo fo th e rdu ckp a tho g en sth a tc anb ep r e s en tdu r ing mu l t ip l ein f e c t ion sw i th DHAV -1 ,DHAV -3 ,andDA s tV -1 ,in c lud ingdu ckp l agu e v i ru s ,av i anin f lu en z av i ru s(H9N2 ) ,N ew c a s t l ed i s e a s e v i ru s ,th ecomp l e t eg enom i c DNAo fdu ckc i r cov i ru s s t r a inF J0601 ,R i em e r e l laana t ip e s t i f e r andE s ch e r i ch ia V IROLOG ICAS IN ICA
L in l inChene ta l
co l i .A l lo fth e s eo th e rp a thog ens amp l e st e s t edn eg a t iv e , ex c ep tinth ec a s eo fadm ix tu r ew i thon e(o r mo r e )o f DHAV -1 ,DHAV -3 ,andDA s tV -1(F igu r e1C) . Ato t a lo f48c l in i c a ll iv e rs amp l e sw i thsu sp e c t ed v i r a lh ep a t i t i sin f e c t ionw e r eth ent e s t edino rd e rtoev a l u a t eth ep e r fo rm an c eo fth e mu l t ip l exRT -PCRa s s ay .In add i t ion ,as imp l exRT -PCRa s s ay ,v i r a lcu l tu r e ,anda n eu t r a l i z a t iont e s tw e r ee a chund e r t ak enonth e48s am p l e s ,a sp r ev iou s lyd e s c r ib ed(Ch enLL ,e ta l . ,2013 ) . Th ep r im e r su s edinth es imp l ex RT -PCRa s s ay sw e r e th es am ea sfo rth e mu l t ip l ex RT -PCRa s s ay . Ov e r a l l , e igh ts amp l e sw e r eid en t ifieda sco in f e c t edw i thDHAV 1 /DHAV -3 ,1s amp l ew a sid en t ifi eda sco in f e c t ed w i th DHAV -1 /DA s tV -1 ,3s amp l e sw e r eid en t i f i eda sco in f e c t ed w i th DHAV -3 /DA s tV -1 ,and36s amp l e sw e r e showntob epo s i t iv efo re i th e r DHAV -1(10s amp l e s ) , DHAV -3(18s amp l e s ) ,o r DA s tV -1(8s amp l e s )a lon e . Nos amp l ew a sfoundtob eco in f e c t edw i tha l lth eth r e e v i ru s e stog e th e r .Th er e su l t sw e r econfi rm edbys imp l ex RT -PCR ,v i r a li so l a t ion ,andan eu t r a l i z a t iont e s t–and w i th100%ag r e em en t . DVHi so fe conom i cimpo r t an c eina l ldu ck r e a r ing r eg ion so fth e wo r ld .InCh in a ,in f e c t ionh a sb e ena s so c i a t ed w i thth r e etyp e so fv i ru s : DHAV -1 , DHAV -3 ,and DA s tV -1(Ch enL ,e ta l . ,2012 ;Ch enLL ,e ta l . ,2013 ; D ingC ,e ta l . ,2007 ;FuY ,e ta l . ,2008 ,2009 ;G aoJ ,e t a l . ,2012 ;W angL ,e ta l . ,2008 ;W angX ,e ta l . ,201 1 ;Xu Q ,e ta l . ,2012 ) . Du ck l ing sin f e c t edbyanyon eo fth e s e th r e ev i ru s e sc anexh ib i ts im i l a rsymp tom sind iv idu a l ly , su cha sasho r tin cub a t ionp e r iod ,sudd enon s e t ,h igh mo r t a l i ty ,op i s tho tonu s ,anden l a r g edl iv e rw i thd i s t in c t e c chymo t i ch emo r rh ag e s . Ap r e sump t iv ed i agno s i so f DVHc anb em ad eb a s edonth eh i s to ryandl e s ion s ,bu t i ti sd i f f i cu l ttoa s c e r t a in wh i chtyp e(o rtyp e s )o fv i ru si sc au s ingth ein f e c t ion .In2008 ,a mu l t ip l exPCR w a sd ev e lop edtod i f f e r en t i a t eb e tw e enth e DHAV -1 and DHAV -3s t r a in s(K im MC ,e ta l . ,2008 ) ;and mo r e r e c en t ly ,w ed ev e lop edanimp rov eddup l ex RT -PCR m e thodth a tf a c i l i t a t e sth er ap idandco s t e f f e c t iv el abo r a to ryd e t e c t iono fm ix edin f e c t ion sc au s edbyd i f f e r en t DHAV -1andDHAV -3s t r a in sindu ck l ing s(Ch enLL ,e t a l . ,2013 ) .N e i th e ro fth e s e mu l t ip l exPCRa s s ay scou ld d e t e c tDA s tV -1 ,how ev e r . Inth ep r e s en ts tudy ,a mu l t ip l ex RT -PCRh a sb e en d ev e lop edfo rth es imu l t an eou sd e t e c t iono f DHAV 1 , DHAV -3 ,and DA s tV -1inc l in i c a lsp e c im en s .T oth e b e s to fou rknow l edg e ,th i si sth ef i r s td e s c r ip t iono f a mu l t ip l ex RT -PCRth a tc and e t e c tth eth r e etyp e so f DVHv i ru s e s .Th e mu l t ip l ex RT -PCR m e thodi s DVH v i ru s sp e c i f i canddo e sno tamp l i fyg enom i c DNAo r RNAf romo th e rdu ckp a thog en s .I t sd e t e c t ionl im i ti s 2 e s t im a t edtob e10pgo fto t a ll iv e rt i s su eRNA ,o r10 cop i e so fe a cho f DHAV -1 , DHAV -3and DA s tV -1v i r a l RNA .Fu r th e rmo r e ,ad i f f e r en t i a ld i agno s i so f DHAV www .v i ros in .o rg
1 , DHAV -3 ,and DA s tV -1 m ayb eob t a in edinas ing l e r e a c t ion w i th inaf ewhou r s , wh e r e a sd i f f e r en t i a t iono f th eth r e ev i ru s e sfo l low edbyc ro s s -n eu t r a l i z a t iont e s t s t ak e ss ev e r a ld ay sa tl e a s t .Th i s mu l t ip l exPCR m e thod i sth e r e fo r ear ap id ,e f f e c t iv eandp r a c t i c a ltoo lfo rth e d i f f e r en t i a ld i agno s i so fm ix edin f e c t ion sw i thth eth r e e typ e so f DVHv i ru s e s ,andfo rth eep id em io log i c a lsu r v e i l l an c eo fDVH . FOOTNOOTES Th i ss tudy w a sfund edbyg r an t sf romth eSh andong Mod e rn Ag r i cu l tu r a lT e chno logy &Indu s t rySy s t em(SDA IT -13 -01 1 -15 ) andth eS c i en c eandT e chno logy Comm i s s iono fSh andong P rov in c e(2010GNC10914 ) ,Ch in a . A l lth eau tho r sd e c l a r eth a tth eyh av enocomp e t ingin t e r e s t .A l l in s t i tu t ion a landn a t ion a lgu id e l in e sfo rth ec a r eandu s eo fl abo r a to ryan im a l sw e r efo l low ed . 1 ,2# 1 ,2 L in l in Ch en1,2#, M ing j i eM a , Ru ihu aZh ang ,Q i an 1 ,2 3 4 1 ,2 Xu , X ingku iS i, Y uW ang, Zh i j ing X i e ,Sh i j in 1 ,2 J i ang
1 .D ep a r tm en to fP r ev en t iv eV e t e r in a ry M ed i c in e , Co l l eg eo f V e t e r in a ry M ed i c in e ,Sh andong Ag r i cu l tu r a l Un iv e r s i ty ,T a i an 271018 ,Ch in a 2 .Sh andongP rov in c i a lK eyL abo r a to ryo fAn im a lB io t e chno logy andD i s e a s eCon t ro landP r ev en t ion ,T a i an271018 ,Ch in a 3 . Depa r tmen to f An ima l Med ic ine , Fo shan Sc ienceand T e chno logyUn iv e r s i ty ,Fo sh an528231 ,Ch in a 4 .D ep a r tm en to fB a s i cM ed i c a lS c i en c e s ,T a i sh an M ed i c a l Co l l eg e ,T a i an271000 ,Ch in a Co r r e spond en c e : Phon e :+86 -538 -8245799 ;F ax :+86 -538 -8245799 . Em a i l :s j j i ang@ sd au . edu . cn # Th e s eau tho r scon t r ibu t edequ a l lytoth i swo rk . Pub l i sh edon l in e :22 M ay2014
REFERENCES Ch enL ,XuQ ,Zh angR ,e ta l .2012 .JV i ro l ,86 :13833 -13834 . Ch enLL ,XuQ ,Zh angRH ,e ta l .2013 .JV i ro lM e thod s ,192 :12 -17 . Ch engAC ,W ang MS ,X in H Y ,e ta l .2009 .J M i c rob io lM e thod s ,77 : 332 -336 . D ingC ,Zh angD .2007 .V i ro logy ,361 :9 -17 . FuY ,P an M ,W angX ,e ta l .2008 .V e tM i c rob io l ,131 :247 -257 . FuY ,P an M ,W angX ,e ta l .2009 .JG enV i ro l ,90 ,1 104 -1 108 . G aoJ ,Ch enJ ,S iX ,e ta l .2012 .V i ro lS in ,27 :248 -253 . Hu angQ ,Y u eH ,Zh angB ,e ta l .2012 .50 :3318 -3323 . K im MC ,KwonYK ,JohSJ ,e ta l .2007 .A v i anD i s ,51 :540 -545 . K im MC ,KwonYK ,JohSJ ,e ta l .2008 .A v i anP a tho l ,37 :171 -177 . W angL ,P an M ,FuY ,e ta l .2008 .V i ru sG en e s ,37 :52 -59 . W angX ,W angY ,X i eX ,e ta l .201 1 .JV i ro lM e thod s ,171 :8 -12 XuQ ,Zh angR ,Ch enL ,e ta l .2012 .JV i ro l ,86 :13848 . Y ang M ,Ch engA ,W ang M ,e ta l .2008 .JV i ro lM e thod s ,153 :55 -60 .
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