Tgp chi Cong ngh? Sinh hoc 12(3): 543-550,2014
iTNG D V N G K Y T H U A T M U L T I P L E X - P C R P H A T H l f N VIBRIO CHOLERAS, VIBRIO PARAHAEMOLYTICUS V A C A C G E N D Q C LlTC C U A C H U N G TlT S A N P H A M T H U Y H A I S A N T U * a i
Truong Huynh Anh V u ' , N g u y i n Ng9c Tudn^
'Trung ldm Dfch vu Phdn tich Thi nghi?m thdnh phd Hd Chi Mmh (CASE)
^Tru&ng Dgi hge Nong Ldm thdnh phd Hd Chi Mmh Ngdy nhgn bdi: 05.8.2014
Ngay nhgn dang: 30.8.2014 T6M TAT
Mgc tieu ciia nghien ciia Id so sdnh kha nang phat hifn Vibrio parahaemolylicus. Vibrio cholerae bing phuong phdp t m y k thdng sii dgng m6i trufmg phan Igp TCBS, CAV (CHROM agar Vibrio) vd PCR. D6ng thdi ling dgng ky thugt m-PCR de phdt hi?n cac gen dgc luc ctxA. rfO\, rfbO\39 cua V. cholerae vd tlh. tdh ciia V. parahaemolylicus Uong thdnh phdm thiiy hai sdn tuoi. Tong so 313 m^u gom fillet ca (126 mlu), tfim (52 mau) vd nghSu (135 mau) duoc thu thgp tgi Trung lam Dich vu Phan ti'ch Thi nghiem a TP. Ho Cht Minh. Ket qua cho thdy ty I? phat hi|n V. parahaemolylicus tren moi trudng TCBS ia 22,0%, tten CAV la 37,7% vd PCR 1^ 43,5%. Tuong hr, ty Ie phat hi^n V cholerae tten moi trudng TCBS la 12,5%, tren CAV \k 24,3% va PCR la 27,5%. E>€ phdt hign cdc gen dpc Igc ciia V. cholerae, m-PCR2 du^c su dgng DNA ly trich tir dung djch tSng sinh ciia cac mau duong tinh vdi V. cholerae (86 mau) va cac chiing V. cholerae phan lap tir TCBS (39 chiing), CAV (76 chiing). Kat qua cho thiy ty I^ phdt hiSn V cholerae 0\IO\39vk V. cholerae aon-0\/non-0139 sinh d$c to td til djch tang sinh cao hon tir cdc chung phan lap; trdi lgi V. cholerae 01/O139 khong sinh dgc to ta tir djch ttog sinh v^ cic chiing phin Idp thi chenh lech nhau khong ddng ke. De phat hien yeu to gay dung huyet ciia V. parahaemolylicus, m-PCR3 dupc sir dgng voi DNA ly trich tir dung dich tang sinh mau (136 mlu), cdc chiing V. p(7raAaemt»/>'r(cw5 tir TCBS (69 chung) va CAV (118 chung) K6t qua cho thdy tj'1^ phdt hi^n gen tdh tir djch ting sinh cao hon cic chung phan Igp, ttong khi do 100% mau tiing sinh lan cdc chiing phan Igp deu sd hihi gen tlh.
Tu khoa: Vibrio cholerae. Vibrio parahaemolylic multiplex-PCR
:, gen doc Igc; torn, fillet ca va ngheu thanh pham tuoi.
DAT VAN DE
An todn ve sinh thuc pham ndi chung vd an todn sdn phdm nu6i trdng thiiy san noi rieng Id vdn d€
quan tam hang ddu ciia nhiSu quoc gia ciing nhu cdc Uij trudng thvc phdm tren thS gicri trong do co Viet Nam. Vi?c sir dyng ciing nhu tieu thy sdn pham thuy hai san khdng an todn thuang gay ra cdc vg ngo ddc nghigm trpng. Nhom Vibrio (V.) dugc biet nhu Id tdc nhdn chinh gay ngp dpc thyc ph^m trong cac san pham thuy hdi san tuoi song. Trong nhihig loai Vibrio gdy b^nh, nguoi ta thuang quan tam dSn V.
cholera. V. parahaemolylicus, V. vulnificus, V.
municus. Trong d6, V. cholerae la loai quan trpng nhat, tuy nhien chi co hai serogroup O l , 0 1 3 9 gdy benh djch td tren nguai. Vi chiing tdn tgi trong mdi trucmg nu6c, ddc biet 6 cac khu vuc gdn ciia song
(Tantillo et al. 2004) nen loai vi khuan nay rat de nhilm vdo cdc dpng vdt thuy hdi sdn. Do do viec an nhiJTig sdn phdm thiiy hai san chua ndu chin Id dieu ki6n de b?nh phdt sinh. Ben cgnh V. chlorae thi thdi gian gdn ddy cdc vy ngd dpc do tieu thy thiiy hdi sdn thucmg lien quan den V. parahaemolylicus. Song khong phai loai V. parahaemolylicus nao ciing gdy b£nh. Trong do chi cd dong mang gen sdn sinh ddc td hemolysin mdi cd khd ndng gay b?nh. Lodi V.
parahaemolylicus gay benh sa hiru mot trong ba gen sdn sinh dpc to hemolysin gdm tdh vd trh md hoa hemolysin ben nhiet (Honda el al. 1988) va tlh ma hoa hemolysin khdng ben nhiet (Taniguchi et al.
1986).
Do do, tdm sodt su hi?n dien ciia cdc lodi vi khuan co khd nang gay ngp dpc thuc phdm cho ngudi tieu thy sdn pham thiiy hdi san tuoi Id vdn de
cdn dat ra thudng xuySn nham gdp phan vao viec bdo v? sire khde cpng ddng. Trong chuang trinh tdm soat do, viec phat hien nhanh va chinh xdc V.
cholerae, V. parahaemolyticus Id mdt yeu can rat cdn thiSt. Myc tieu ciia nghidn cihi nay la so sdnh kha nang phat hien V. parahaemolylicus, V. cholerae bdng phuong phap truyen thdng sii dyng moi trudng phan Igp TCBS (Thiosulfate Citrate Bile Saccharose), CAV (CHROM agar Vibrio) va phuong phdp PCR (Polymerase Cham Reaction). Mdt khac, sir dung ky thugt m-PCR de phdt hien cac gen dpc luc ciia vi khuan V. parahaemolylicus vd V. cholerae trong sdn phdm thuy hai san.
NGUYEN LIEU VA PHU'ONG PHAP M§u hdi sdn
313 mdu thdnh phdm thuy hdi sdn tuai hogc ddng lanh thdnh phdm duoc thu thgp tai Trung tdm Dich vy Phan tich Thi nghiem d TP. Ho Chi Minh.
Phdn Idp V. cholerae, V. Parahaemolyticus Can 25 g mdu cho vdo tui PE vd triing. Sau do, bd sung vao 225 ml mdi trudng tSng sinh ASPW (Alkaline Salme Peptone Water). U mlu d nhiet do 37''C trong 18 gid doi vdi cdc san pham dong lanh sau hogc d 41,5"C trong 6 gid ddi vdi san pham tuai.
ChuySn I ml dich cdy cho vdo dng nghiem chiia 10 ml mdi trudng tang sinh ASPW, li d 41,5''C trong 6 gid. Diing que cay vdng Idy dich cdy thu dupc trong ASPW, ciy len thgch TCBS vd CAV, u d 37"C trong 24 gid. Tii moi trudng thgch TCBS, chpn khodng 3 -
Truong Huynh Anh Vu & Nguyen Ngpc 1 ^ 5 khudn Igc di^n hinh cua V. cholerae hogc K parahaemolyticus dk cdy chuydn. Cay tung khnb Igc da chon len hk mat nghieng ciia thgch NA (Nutrient Agar), d 37°C trong 24 gid dk thii sinh bdi
C h u ^ n bi DNA khuon ^]
Chiing tdi ti^n hdnh ly trich DNA ctii V. cholerae vd V. parahaemolylicus phan Igp ilujc nhu sau:
Ddi vdi dich t^ng sinh, lay I ml dung dich tdng sinh cho vao Eppendorf. Doi vdi cac chiing da ki^
tra sinh hda, Idy mpt sd khudn Igc tren thach NA cho vao Eppendorf chua sin 1 ml nude cat 2 lan vo triing. Ly tdm 6000 vdng/phiit trong 5 phiit 6 nhi^
dd phdng. Logi bo dich ndi, giii lgi phdn sinh khoi tv d day, them 1 ml nude cdt 2 ldn. Vortex mgnh dJ sinh khdi dugc tron deu. Ly tdm 6000 vong/pblt trong 5 phut d nhidt dp phdng. Loai bd djch ndi, gift lgi phdn smh khdi tu d day. Bo sung 500 pl nude dt vo tning, vortex mgnh dk huydn phii deu. D^
Eppendorf vao mdy li lac d 100"C/15 phut Ly (am 13000 vdng/phiit trong 5 phiit d nhiet dp phdng. liy phdn dich trong ben tren de tien hdnh phan 6ng PCR.
Phdt hifn V. cholerae, V. parahaemolyticus, gen CtxA, rfb ciia V. cholerae va gen tlh, tdh cda V. parahaemolyticus
Mdi cho phdn ihig
Cdc dogn mdi cho phdn iing m-PCR (multiplex- PCR) dd phdt hien V. cholerae, V parahaemofylics^
gen CtxA. ifOl. rfbO\39 ciia V. cholerae va tlh, 1^
ciia V. parahaemolyticus dupc trinh bdy d Bdng I.
Bing 1 . M 6 I SCP dgng phat hign V. cholerae, V. parahaemolyticus vi c i c gen a$c lyc Kichca*
san phSm Ph&n i m g Ngudn
V. parahaemolyticus Vp.fiaE-79f WJ.flaE-934R
AAG ACC TCA ACT GGC GGT A GAA GTG TTA GTG ATC GOG AGA GT GCA GCT GAT CAA AAC GTT GAG T ATT ATC GAT CGT GCC ACT CAC CTC AGA CGG GAT TTG TTA GGC ACG TCT ATC TCT GTA GCC COT ATT ACG
0139/ft-F 0139/flj-R
AGC CTC TTT ATT ACG GGT GG GTC AAA CCC GAT CGT AAA GG
(dh-F
tdh-R GTA AAG GTC TCT GAC TTT TGG AC TGG AAT AGA ACC TTG ATC TTC ACC
Tgp chi Cdng nghi Sinh hge 12(3): 543-550,2014 Thinh phan phan ihig P C R
G6m 2 lU AmpHTaq Gold; 0,2 mM dNTP; 1,5 mM MgCb; ddm IX; 0,5 pl mdi dogn mdi (ndng dp 0,625 pM); 5 pl DNA mau xet nghidm vd nude cit khli ion hai ldn vira du den thd tich 25 ^1.
Quy trinh nhiet cua phdn iing m - P C R l : 93°C/15 phiit (I chu k^'); 92°C/40 gidy, 57°C/1 phiit, 72''C/1,5 phiit (35 chu kS-); 72°C/7 phut (1 chu kJ-).
Quy trinh nhiet cua phan iing m-PCR2: 95°C/5 phiit (1 chu ky); 95°C/30 gidy, 50"C/30 giay, 72°C/I phut (40 chu ky); 7 2 ' ' a 6 phiit (1 chu k>').
Quy trinh nhi?t cua phan ung m-PCR3: 94°C/3 phut (1 chu ky); 94°C/1 phut, 58"C/1 phiit, 72°C/I phut (30 chu ky); 72''C/5 phiit (I chu ky).
Phdn tich kit qud tren di^n di do
10 pl sdn phdm PCR trdn vdi 2 pl loading dye dupc di^n di trdn gel agarose 1,5% c6 chiia 1 fig/ml ethidium bromide trong TBE. DNA chuan ciing dupc di?n di dong thdi. Thdi gian dien di Id 35 - 40 phiit d 100 V vd 100 mA bdng b l dien di ngang (HR - 2025, IBl). Sau do chup hinh gei vdi tia UV bdng mdy chyp gel Ingemus, phdn m^m GenSnap, SynGen. Cdc bdng DNA dugc xdc dinh bang cdch so vdi cac bdng ciia ddi chiing duoTig vd DNA chuan 100bp(GoldBio,USA).
Vi khudn doi chung duong gom V. cholerae. V.
parahaemolyticus, V. cholerae 0 1 , V cholerae 0139 dugc cimg cap bdi Trung tdm Dich vy Phan tich Thi nghigm TP H6 Chi Minh vd Vifn Pasteur TP HoChiMinh.
Xir ly so lieu
So Ueu dugc xii ly vdi su ho trg ciia phan mem Excel 2010. So sanh cac ty le bdng trde nghidm khi binh phuang (5^).
KET QUA
Phdt hi£n V. parahaemolyticus, V. cholerae nudi c i y trfin moi trirdng TCBS va CAV
Moi trudng TCBS vd CAV diing dk phdn Igp V.
parahaemolytieus, V. cholerae trong cdc mdu khdo sdt Tuy nhien, ty 1? phdt hien V. parahaemolyticus, V.
cholerae giua hai moi trudng tren la khac nhau. V.
parahaemolyticus trong cac mau hdi san dugc phdt hifn bdng nuoi cdy tren moi trudng CAV cao hon so vdi mdi tnrdng TCBS (37,7% so vdi 22,0%) (Bang 2). Ddi vdi tung loai mau, ty Id phdt hien V. parahaemolyticus ciing rat khde nhau. Trong do ty le phdt hipn V.
parahaemolyticus a mdu ngheu cao hon so cac mdu khac (TCBS: 31,1%, CAV: 52,6%). Tuong ty, tren mdi trudng CAV ty 1? phat hien V cholerae cao htm so vdi mdi ttudng TCBS (24,3%). Mat khde, ddi vdi timg logi mdu khdo sdt, t^ Ip nhiem V. cholerae ciing khde nhau.
Ddng quan tam hem Id mau nghdu cd mirc dp nhilm V.
cholerae cao hon cdc Igai mdu cdn lai (TCBS: 20,0%, CAV: 36,3%).
Trde nghiem x^ dugc sii dyng dd so sdnh cdc ty Ig.
Ket qua cho thdy ty Id K parahaemolyticus, V. cholerae dugc phat hien tren mdi trudng CAV cao hon nhieu so vdi moi ttudng TCBS (P < 0,05). Nhu vay, moi trudng CAV la mdi trudng Ihich hpp de phdn l ^ va dinh danh V. cholerae, V. parahaemolyticus.
Bdng 2. So sSnh ty Ig phat hign V. parahaemolyticus Logl moi tnp£mg
Chi tieu Logi mdu Filleted (126) Tflm(52) Ngheu (135) Teng cdng
TCBS V. parahaemolvticus
n 24 3 42 69
%
19,0 5.8 31,1 22,0
, V cholerae Q\Oa
V. cholerae n 8 4 27 39
%
6.3 7.7 20.0 12,5
TCBS va CAV CAV V. parahaemolyticus
n 32 15 71 118
%
25,4 28,8 52,6 37,7
V. cholerae n % 16 12.7 11 21.2 49 36.3 76 24.3 n: S6 mSu duong tinh
So sdnh k^t qud phat hifn K cholerae, V.
parahaemolyticus bdng phumig phdp truyin th6ng vdPCR
Trong nghien ciru ndy, cdc mdu khdo sdt duong tinh vdi V. cholerae, V. parahaemolylicus dugc the hign d hinh I. Phuong phap m-PCR 1 dugc dimg de djnh danh V. cholerae, V. parahaemolyticus cd ty le
phdt hipn cao hon phuong phap truyen thdng d cd hai mdi trudng phan lap TCBS va CAV. Ddi vdi V.
cholerae ty- Id phdt hign ciia m-PCRl la 27,5% so vdi 12,5% tren moi ttudi^ TCBS va 24,3% fren CAV (Bang 3). Tuong tu, ty le phat hien V parahaemolyticus cua m-PCRI la 43,5% so vdi 22,0%
tten mdi ttirdng TCBS va 37,7% tren CAV (Bdng 4).
Truong Huynh Anh Vu & Nguyin Ngpc "^
Bang 3. So sanh ty i^ phat Phutyng phap Loai mau Fillet cd (126) Tdm (52) Nqheu (135) Tong cpng
n: So mau ducmg tinh
Bang 4. So s ^ h ty le phat P h m m g phap Loai mau Fillet C3 (126) Tdm (52) Ngheu (135) l o n g cpng
hien V. cholerae giOa
hien V n 20 11 55 86
m-PCR-
'. parahaemotyti
n 43 18 75 136
m - P C R I
phucmg phap tmy^n th6ng va m - P C R l 1
%
15,9 21,2 40,7 27,5
CUS giua
%
34,1 34,6 55,6 43,5
n 8 4 27 39
TCBS
Truydn t h i n g
% n 6.3 16 7.7 11 20.0 49 12,5 76
phucrng p h i p tnjyen Hidng v i m - P C R l
n 24 3 42 69
TCBS
Truven thong
% n 19.0 32
5.8 15 31,1 71 22,0 118 CAV
CAV
1
d%^
12.7 *"
21,2 ' 36.3 24.3 -
%
25,4 26,8 52,6 37,7 n. S6 miu duong tinh
Xet tren ddi tugng mdu khdo sdt, m - P C R l cd ty Ip phat hien dugc V. cholerae cao hon nhidu so vdi phuang phap truyen thdng sir dyng mdi trudng TCBS (fillet ca: 15,9% so vdi 6,3%; tom: 21,2% so vdi 7,7%; ngheu: 40,7% so vdi 20,0%). Tuy nhien, ty Id nay chenh lech khong dang ke so vdi
phuang phdp truydn thdng sir dyng mdi tnrong CAV (fillet cd: 15,9% so vdi 12,7%; nghdu: 40,7%
so vdi 36,3%) (Bang 3). Ty If phdt hifn ¥.
parahaemolyticus trdn nghdu cao ban tdm vd fillet ca rat rd ret trong cd hai phuang phap sii dgng (Bang 4).
- 897 (VcsodB) -248(Vp.fflsE)
Hnh 1. K4t qua phat hi$n V. cholerae. V. parahaemolyticus cua mpt so miu khao s^t bSng phdn irng m-PCR1
Phat hifn cdc gen ddc l|rc cua V. cholerae vk V. parahaemolyticus
Phdt hien cdc gen dgc lire cda V. cholerae Sau khi phdt hien V. cholerae ttong m l u khdo sat bang phdn iing m - P C R l , cdc doan mdi etxA, rfbOl vd rfbO\39 dugc sir dyng trong phdn ung m - PCR2. Phan iing m-PCR2 dugc thuc hidn vdi DNA Iy ttich tli hai nguon gdc kbdc nhau: 86 mdu DNA ly trich tir dung dich tang sinh ciia cdc mau duang tinh vdi V. cholerae d m-PCRI va 115 mdu DNA dugc ly trich tir 115 chiing V. cholerae (39 chiing trdn mdi trudng TCBS vd 76 chiing tten CAV) dS dugc dinh
danh bang vifc thur smh hda va khdng djnh lgi b ^ m - P C R I . Cac m l u khdo sat phat hifn cdc gen dpc luc ciia V. cholerae ddi vdi phdn iing m-PCR2 <to?c thd hien d Hinh 2.
Ket qud Bang 5 cho thdy ty If V. cholerat 0 1 / 0 1 3 9 sinh dgc td td tir DNA ciia dich tdng siiA mdu Id 16,28% so vdi cac chiing phan Idp tren TC!IBS la 7,69% vd tren CAV la 7,89%; ty If nhdra Y.
cholerae n o n - O l / n o n - 0 1 3 9 sinh dgc td td tir DNA cua dich tang smh m l u la 5,81% cao hon tu chdDg phan lap (0 va 2,63%); tt^i lai V. cholerae OM0\'i9 khdng sinh dpc to ta tii DNA ciia djch ting sinh U 8,14% tuong duong vdi DNA tir chiing phdn Igp trio
Tgp chi Cong ngh? Sinh hoc 12(3): 543-550,2014 TCBS (7,69%) nhung thap hon DNA tii chung phdn Igp tren CAV (10,53%). N h u vay DNA taeh chidt tir dung dich tang sinh m l u da sd hiiu nhidu gen ddc luc vdi ty If cao ban so vdi DNA cua cdc chung phan Igp. Dieu nay cho phep ta nhdn dinh rang mdi mau xdt nghifm da duang tinh vdi m - P C R l thi DNA tdeh chift tryc tidp tu dung dich tang sinh ASPW da bao ham khd nhieu sd te bao cac nhdm V. cholerae
trong mau, trong khi do DNA ciia moi chimg V cholerae sau khi tang sinh vin khong thf dai difn cho qudn the nhdm vi khudn ndy trong mau xet nghiem. Do do de tdm sodt cdc yeu to dpc luc ciia V.
cholerae ttong san pham thuy hdi san, cdc phdng thi nghifm giam sat nen sir dung DNA tdeh chidt tryc tiep tu dung dich tang sinh mlu xet nghiem hem Id chgn khudn lac.
B ^ g 5. Ket qud phdt hien gen
Cac gen d c h phat hign ctx^*rfbo^ +/ftoi39
c(xA + rfbOl c(xA + ra)0139
/ f t O I ffl)0139
c(xA
dpc l y c cua V cholerae Djch tang s i n h
(n = 86 mSu) n 2 5 7 3 4 5
%
2,33 5,81 8.14 3,49 4,65 5,81
(n n 0 0 3 1 2 0
TCBS
= 39 chiing)
%
0,00 0,00 7,69 2,56 5.13 0,00
(n 0 2 4 3 5 2
CAV
= 76 c h i i n g )
%
0,00 2.63 5.26 3,95 6.56 2,63
Hinh 2. Ket qua phat hi^n cac gen flpc lyc cua V. cholerae cua mdt s6 mlu khdo sdt bang phSn iJng m-PCR2
Phdt hifn cdc gen dgc lire cda V. parahaemolyticus Sau khi phdt hien V. parahaemolyticus ttong mdu khao sat bang phan iing m - P C R l , cdc dogn mdi tdh va tlh dugc sii dyng trong phan iing m - P C R 3 . Phdn ling m-PCR3 dugc thyc hien vdi DNA ly trich tii hai ngudn gdc khac nhau: 136 mdu DNA ly trich tir dung dich tang sinh ciia cac mau duang tinh vdi V. parahaemolytieus a m - P C R l va 187 mdu DNA dugc ly trich tir 187 chiing V parahaemolyticus (69 chiing tu mdi trutmg TCBS va 118 chiing tir moi trudng CAV) da dugc dinh danh bdng sinh hda vd khdng djnh lai bdng m - P C R I . Cdc gen dpc luc cua V. parahaemolyticus dugc phat hien trong m§t sd mdu khdo sdt bdng phdn ung m-PCR3 dupc the hifn d Hinh 3.
Ty If [Mt hifn gen idh tii DNA cua djch tang smh mlu Id 25,74% cao ban cdc chiing phan Igp (20,29%
tii TCBS va 16,10% tir CAV), trong khi do 100%
mdu tang sinh lan cac chung phan lap deu sd hiiu gen tlh (Bdng 6).
Nhu tten da de cap, m-PCR3 dfu phat hifn 100% gen dgc luc tlh cua V. parahaemolyticus duong tinh vdi m - P C R l vd sinh hda. Tdt cd cdc chiing V. parahaemolylicus ttong phgm vi nghien Cliu nay deu phat hien cd gen tlh.
Ket qua Bdng 6 cho thay sir dyng DNA ly trich true tiep tii dich tang sinh phat hien ddng thdi hai gen tlh va tdh cao han so vdi cdc chiing dugc dinh danh bdng sinh hda. So sanh khd nang phdt hldn ddng thdi hai gen tlh vd tdh tir hai mdi trudng phan lap thi mdi trudng CAV lai thdp han mdi trudng TCBS (16,10% so vdi 20,29%).
Tnrong Huynh Anh Vu & Nguyfn Nggc Tuan Bang 6. K^t quaphat hien gen 3^c \gc c
tlh + tdh tlh
:ua V. parahaemolyticus Djch tang sinh
(n = 136 m l u ) n % 35 25,74 136 100
TCBS (n = 69 c h i n q ) n % 14 20.29 69 100
(n = n 19 118
t
CAV - 118 Chung) '.
16,10 "
% ~
m .
-45D(i;/i) -270(fd/i)
Hinh 3. Kit quS phat hien cac gen dpc lyc ciia V parahaemolyticus cua mOt s6 miu khao sSt bing phan irng m-PCR3
THAO LUAN
Phdt hifn V. cholerae vd cdc gen ddc lire Vdi phuang phap truyen thdng su dyng dong thdi 2 logi mdi trudng phan Igp khac nhau, chiing tdi da phan Igp dugc 115 chiing V. cholerae. Sau dd, chiing dupc khang dinh lgi bdng m-PCRI vd cho thdy 100% chiing phan lap la V. cholerae. Cho nen, phuong phap nudi cay truyen thdng vdn dugc sii dung rpng rai de djnh danh V. cholerae. Tuy nhifn phuong phdp PCR nhanh hon, nhgy han vd ty If phdt hien ludn cao hon phuong phdp truyfn thdng d ca hai mdi tnrdng phdn lap (27,5% so vdi 12,5% tren mdi ttirdng TCBS vd 24,3% tren CAV). Kdt qud ndy phii hgp vdi nhgn dinh ciia Nguyen Thi Xudn Trang vd Nguydn Nggc Tudn (2012). Ngoai ra, Wang vd cs (2010) cho bift ty le phat hien V. cholerae trong mau nude d cdc viing cira sdng Trung Qude bang phuang phap PCR cao hem so vdi phuang phap truyfn thdng (29,5% so vdi 10,3%).
Nhiing nghifn ciiu tnrdc ddy cho ket qua tuong ty ve khd nang dinh danh V. cholerae. Choopun vd ddng tdc gia (2002) sir dung 13 phdn dng sinh hda de dinh danh V. cholerae tir mau nude va khang dinh lai bdng phuong phap PCR, kdt qua cho thdy ttong 301 chiing V. cholerae dinh danh bang sinh hda, chi c6 3 chimg am tinh vdi PCR. Trong nghien ciiu ndy su hien difn cua V. cholerae ttong mdu hai san bdng phuang phdp truyen thdng vd khdng dinh khudn Igc bang PCR ldn lugt la 12,5% trdn radi trudng TCBS
vd 24,3% tren CAV thap hon ket qua ma Chitov vi ddng tdc gid (2009) la 35,9%.
Su hifn difn cac gen dpc Igc ciia V. cholerae tir dich tdng sinh cao hon so vdi cac chiing phan l$p tii mdi tmdng TCBS va CAV (Bang 5). L^ do Id PCR c6 khd nang phdt hifn cac dong V. cholerae cd khd naug tdn tgi trdn mdi trudng tang smh nhung khong kha ning phdt tridn trfn moi trudng phan Igp. Ben cgnh dd, vfi cdc chiing V. cholerae sdng trong mdi trudng ty nhifti thi vifc phan lap bang cdc phuang phdp nudi cay thdng thudng Id rdt khd khan (Nguyfn Lmh Todn, 2011) y[
chiing cd thd tdn tai nhung khdng phat trien Iren moi tnrdng nudi cay (Aulet vd cs, 2007). Bdn cgnh d6, Aulet va cs (2007) da chiing minh rang tdt cd cdc mSu duang tinh vdi gen dpc lyc ctxA va tcpA ciia V.
cholerae bdng phuong phdp PCR dfu khong phan % duac bdng phuang phdp nudi cdy tmyf n thong.
Phdt hifn K parahaemolyticus vd cdc gen d§c lifC Kdt qua nghien ciiu cho thdy sii dgng phuong phdp PCR de tdm sodt su hien difn ciia V.
parahaemolytieus cho kft qua chinh xac, lift kifm chi phi vd thdi gian so vdi phuong phdp nudi caiy truyen thdng tren ddi tugng mlu da khdo sdt. Su dyng ky thugt nudi cdy va thu sinh hda de dinh danh V. parahaemolyticus la khdng dang tin cay (Croci ei al, 2002). Khi phdn lap trfn moi tnrdng TCBS, khudn Igc V. parahaemolyticus rat khd phdn bift "A nd bi mdu vang cua khuan Igc V. cholerae khuich tdn, khudn Igc cua V. vulnificus cung mau xanh (Wch 548
Tgp chi Cdng ngh? Smh hge 12(3): 543-550, 2014 thudc be hon) ndn rat de bi nhdm Idn (Hara-Kudo et al, 2001). Theo Raghunath vd ddng tac gid (2008) da ghl nhan ty le m l u nhifm V. parahaemolyticus mang gen dgc lyc tdh tang dan theo thdi gian nudi cdy tang sinh lln lugt la 5,2%, 10,3% vd 13,8% sau 0 gid, 6 gid vd 18 gid. Didu nay ciing phii hgp vdi ket qua nghien ciru khi phat hifn ddng thdi hai gen tlh vd idh ciia DNA ly trich true tidp tir dich tang smh cao ban so vdi cdc chiing dugc dinh danh bang sinh hda (Bang 6). Vi vgy, giai doan nudi cay tang sinh Id rat cdn thiet cho viec phat hien V. parahaemolyticus va cac gen doc lyc ciia chiing bang ky thudt PCR ttong nhihig mau hai san.
KET LUAN
Ty If nhiem V. cholerae trong mau fillet cd, tdm va ngheu la 27,5% thap han V. parahaemolyticus Id 43,5%. Sy hifn difn ciia V. cholerae 0 1 / 0 1 3 9 va V.
cholerae non-01/non-0139 mang gen san sinh ddc td td ttong mdu khao sat thap (lan lugt la 16,28% va 5,81%). Ty If phat hifn ddng thdi hai gen ddc luc tlh, tdh ciia V. parahaemolyticus Id 25,74% vd 100%
gen ddc luc tlh dugc phdt hifn.
TAI LIEU THAM K H A O
Aulet O, Silva C, Fraga G, Pichel M, Cangemi R, Gaudioso C, Porcel N, Jure A, Cecilia M, Binsztein N (2007). Detection of viable and viable nonculturable V cholerae 01 through cultures and immunofluorescence in the Tucuman rivers, Argentina Revista da Sociedade Brasileira de Medicina Tropical 40 (4): 384-390.
Chitov T, Wongdao S, Thatum W, Puprae T, Sisuwan P (2009). Occurrence of potentially pathogenic Vibrio species m raw, processed, and ready-to—eat seafood and seafood products. Mje/o/nf. y. Sci. Tech3il). 88-98.
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Honda T, Ni Y, Miwatani T (1988) Purification and characterizahon of a hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus and related to the thermostable direct hemolysin. Infect. Immun 56: 961-965
Nguyen Lmh Toan (2011). Nghien ciiu ling dung kJ thuat PCR da moi phat hien Vibrio cholerae gay h6nh ta. Tgp chi Yhoc thuc hanh 748 (1): 120-123,
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Tnrong Huynh Anh Vu & Nguyfn Ngpc Tufe
A P P L I C A T I O N O F M U L T I P L E X - P C R T E C H N I Q U E F O R D E T E C T I O N O F VIBRIO CHOLERAE, VIBRIO PARAHAEMOLYTICUS A N D T H E I R V I R U L E N C E G E N E S FROM F R E S H S E A F O O D
Truong Huynh Anh Vu''-, Nguyen Ngoc Tuan^
Center ofAnalitical Services and Experimentation HCMC, CASE Nong Lam University
SUMMARY
The objective of this study were to compare the ability to detect V. cholerae and V. parahaemolylicus by conventional methods using TCBS, CAV medium and PCR method; and also to identify the presence of virulence genes of V. cholerae, V. parahaemoljticus from iresh seafood by using m-PCR technique. A total of 313 samples (126 from fillet fish, 52 from shrimp and 135 from clam) were collected at Center of Analytical Services and Experimentation in HCMC (CASE) The results was showed diat the frequency of V parahaemolylicus was 22.0% in TCBS medium, 37.7% in CAV and 43.5% by PCR method. Similar, the frequency of V cholerae was 12.5% in TCBS, 24.3% in CAV and 27.5% by PCR method. In order to identify cholera toxin of V cholerae, m-PCR2 was performed with the exfracted DNA from enrichment broth of 86 samples, 39 isolates of V. cholerae in TCBS and 76 isolates in CAV. The result was showed that the ftequency of V cholerae OI/0139 serogroup and V. cholerae non-Ol/non-0139 possessed cholera toxin from the enrichment broth were higher than from the isolates in TCBS and CAV, in conttast, the frequency of V.
cholerae 01/0139 serogroup non-camed cholera toxin from the enrichment broth and from the isolates in TCBS and CAV was non-significantly difference. To identify the hemolytic gene of V parahaemolyticus, m- PCR3 was performed with the extracted DNA from enrichment broth of 136 samples, 69 isolates of V.
parahaemolylicus itom TCBS and 118 isolates from CAV. The result was showed that the ftequency oi tdh gene from the ennchment broth higher than from the isolates, 100% the enriched samples and isolates were detected tlh gene.
Keywords: Vibrio cholerae. Vibrio parahaemolyticus. virulence gene; fresh shrimp, fish fillet and clam product. multiplex-PCR.
• Author for correspondence: E-mail: truonshuvnhanhvulfl} yahoo, com 550
