KHOA HQC KY THUAT THU Y TAP XIX SO 1 - 2012
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THIET LAP PHl/ONG PHAP PCR DE XAC DjNH VI KHUAN VEROTOXIGENIC ECOU (VTEC) TRONG THIT
Nguyin Thi Thanh Thuy', DdNgpc ThAy^
vd Nguyin Bd Hiin^
T6M TAT
Phuorng phdf) PCR phiie vdi 3 lo^ii c?lp m6i (VTl, VT2 vd eae), cdc dilu kifn phdn umg nhu dd duge thilt lap va chuin hda trong nghien ciru ndy cd thl dugc dp dyng dl xdc djnh vi khuin Verotoxigenic Escherichia coli (VTEC).
Tir khoa: Verotoxigenic Escherichia coli (VTEC), PCR , Xdc djnh
A study on PCR method to determine Verotoxigenic E.coli bacteria in meat
Nguyen Thl Thanh Thuy, Do Ngoc Thuy and Nguyen Ba Hien
SUMIMARY
Complex PCR method with three kinds of primer pairs (VTl, VT2 and eae), the reaction conditions as established and standardized in this study can be applied to determine the Verotoxigenic Escherichia coli bacteria (VTEC).
Key woiris: Verotoxigenic Escherichia coli (VTEC), PCR , Determination.
LDATVANDfe
PCR (Polymerase Chain Reaction) la mpt ky thuat nhdm khuech dai mpt doan DNA ddc hifu nhd 1 cap mdi. M6i su dyng trong PCR thudng la mpt cdp oligonucleotide, dai Idioang 15-30 nucleotide. Thanh phin ciia PCR g6m: doan DNA dieh, cdc m6i co trinh tu b6 sung vdi DNA dich, hon hgp ciia 4 loai base va mpt DNA- polymerase phii hgp.
Qua trinh khuich dai la mpt sy l^p lai cac chu ky eua tdng va gidm nhift dp. Moi chu ky g6m 3 bude: dan xodn sgi DNA k6p, cdc mbi bit cap vdi tumg sgi DNA dom, tdng hgp sgi DNA mdi nhd DNA-polymerase. Theo dd, so
^Nghien cijru sinh Vi?n sau dai hpc, D?ii hpc Nong K i i ^ p Ha NOi.
Vien thu y.
FKhoa thu y, Dai hpc Nong nghi?p Ha N0i.
lugng DNA tao ra tdng len 2" vdi n la sd chu ky (Saikivacs, 1987).
Mpt trong nhumg img dyng ciia PCR la xac djnh chfnh xac vi khuin. Day la phuang phdp nhanh, d$c hifu, nh^y va ty dpng trong vifc xac djnh cdc mim bfnh la vi sinh v^t. Nhilu tdc gid da sur dyng thdnh cdng phuang phdp nay trong xdc djnh gen vt cua VTEC. Phuang phdp PCR xdc djnh gen vt cd thl phdt hifn dupe dudi 10 VTEC trong tong lO' eoliform la mpt phuang phdp phii hgp de phdt hifn VTEC trong mau thuc pham nghi ngd.
Nhu mpt phuang phdp chin dodn, PCR da dugc phdt triln dl xdc djnh gen vt d E.coli phan l$p tir cdc mlu bfnh phim, thyc pham va phan.
Ddc bift ky thuat PCR phiic la mpt chin dodn cd gia trj djch ti hpe (Nguyfn Binh Minh, 2004).
Nhd ky thuat nay cd thl tiln hanh ddng thdi s6 lupmg ldn mau, tilt kifm thdi gian va sinh phim.
KHOA HQC KY THUAT THU Y T^P XIX S6 1 - 2012 II. NGUVfeN U$U vA PHUOfNG P H A P
2.1. Nguyen lifu
Miu thjt edc lo^i (bd, lgn, gd) dd dugc xdc djnh la am tinh vdi VTEC frong phdn img PCR dl tiln hdnh gay nhilm vdi edc chung vi khuin doi chimg duang.
Cac chiing vi khuin d6i chimg duong vd dm do Vifn nghien curu thu y (NVRI) Ba Lan; Phdng thi nghifm tham chilu E.coli Tdy Ban Nha (Laboratorio de Refereneia de E.coli LREC);
Phdng thi nghifm tham chilu vi khuin E.coli ciia Oil Canada (OIE Reference Laboratory for Escherichia coli) cung cip.
C ac lo^i mdi trudng, hda chit dimg trong nudi cay. phdn i^p vd gidm djnh vi khuin E.coli.
M6i trudng, hda chit diing trong phdn img PCR vd difn di sdn phim PCR do hdng Fermentas sdn xuit g6m cd: AMP x 5 buffer, dNTP stock, 50 x TAE, Taq-DNA polymerase.
Primer, Gel loading buffer, Ethidium bromide.
Cac lo^i dyng cy, trang thilt bj mdy mdc phdng thi nghifm.
2.2. Pbinmg phdp
2.2.1. Phuorng phdp tien hdnh phdn Irng PCR PCR dom mdi: moi phdn umg PCR sg dugc thuc hifn vdi sy tham gia eua mpt c$p m6i d$c hifu dl phdt hifn mpt gen nhit djnh (VTl, VT2 hodc eae).
PCR phirc (da mdi): sir dyng nhilu e$p moi trong mpt phdn img PCR dl phdt hifn m^t s6 gen can thilt. Trong nghien curu ndy Id edc gen VTl, VT2 va eae.
Xir ly DNA mlu: DNA mSu dugc tdch ehilt bdng phuang phdp s6c nhift nhu sau: 1ml canh khuin dugc dun d 100°C/30 phiit, ddng l^uih d 20''C/qua dem, ly tam 13.000 vdng/phut trong 10 phiit. Chit trong d trfn dugc tdch nfng vd sCr dyng lam DNA mau.
2.2.2. Phutmg phdp xdc dinh d0 d^c hi^u cua phdn ung PCR
Dp ddc hifu cua phdn iimg PCR dugc xdc djnh bdng cdch kilm tra vdi 10 chung VTEC d6i
chirng duong vd 10 chiing d6i chiimg dm vdi cdc thdng tin vl d^c tfnh ciia mOt s6 ylu t6 gdy bfnh dd dugc c6ng b6, sir dyng phdn img PCR phiic.
2.2.3. Phutmg phdp xdc dinh dp nhgy cua phdn ung PCR
Tiln hdnh ciy I khuin l^c ciia m5i chung vi khuin vdo Ip dyng 20ml mdi trudng (LB, mTSB vd BHI). sau 20 gid ii d 37°C, tiln hdnh pha lodng canh khuin theo co s6 10 dk dugc cdc ning dO pha lodng thfch hgp (lO', 10•^...10"''), dim s6 trfn mdi trudng th^ch mdu, d6ng thdi tiln hdnh cdc bude tdch DNA miu tir tit cd cdc ning df pha lodng (kl cd canh khuin ban diu) dl diing cho phdn img PCR.
III. Kh QUA vA THAO LUAN
Vdi nhOmg trudng hgp bfnh d ngudi vd dpng
\^l cd lien quan tdi VTEC, trong dd cd cd nhihig bfnh gdy nguy hilm tdi tinh m ^ g con ngudi nhu hpi chumg huylt nif u (HUS), thyc tl ddi hdi phdi cd mOt phuang phdp nh^y vd d^c hifu nhdm phdt hifn vi khuin ndy. Trong dd, PCR da vd dang dugc ddnh gid id phuang phdp chinh xdc, nhanh va tifn dyng nhit dl phdt hifn cdc miu thyc phim ho$c mlu phdn cd chura VTEC thdng qua xdc djnh gen quy djnh cdc ylu to dpe lyc cua vi khuin ndy (Paton A.W. vd J.C.Paton, 2002).
3.1. Thilt l$p, chuin hda phmmg phdp PCR Trong nghien curu ndy, chung tdi da xdy dyng mOt phuong phdp da m6i nhdm xdc djnh 3 lo^i gen dpe lyc ciia cdc chung VTEC doi chimg, tir dd dp dyng d6i vdi cdc ehiing phdn l|ip dugc. Thi nghifn tiln hdnh vdi 3 chiing VTEC, gom 2 chiing ddi chiing duong (FD523, FD636) vd 1 chung d6i chumg Sm (C-600). DNA mlu la mft lugng nhd khuin l^c ciia vi khuin dd dugc nuoi ciy trfn dia th?ich mdu cihi (37°C/24 gid).
Mb\ ciia phdn umg dirge thilt kl dya vao chuSi gen md hod sinh t6ng hgp protein ciia dfc t6 VTl, VT2 vd protein intimin quy djnh d§c tfnh xam nh|ip va bdm dinh cua VTEC. Trinh ty cac m6i vd cdc sdn phim tuomg umg cua chiing tao ra dugc trinh bdy d bdng 1.
KHOA HQC KY THUAT THU Y TAP XIX SO 1 2012
Bang 1. Trinh ty mdi dCing d i xdc djnh cdc gen VTl, VT2 vd eae Gen
dich VT1 VT2 Eae
Ky hifu primers VT1-F VT1-R VT2-F VT2-R eae-F eae-R
ChuSi primers (5" - 3')
5' - GAG TTA ATG TGG TGG CGA AG - 3' 5' - CTG CTA ATA GTT CTG CGC ATG - 3' 5' - CTT CGG TAT CCT ATT CCC GG - 3' 5' - GGA TGC ATC TCT GGT CAT TG - 3' 5' - ACG TTG CAG CAT GGG TAA CTC - 3' 5' - GAT CGG CAA CAG TTT CAC CTG - 3'
Kich cd sdn phim (bp)
894 481 816
Bang 2. Thdnh phdn cdc chit trong phdn iimg PCR dung d i xdc djnh cdc gen VTl, VT2 vd eae Thdnh phin
Mii xudi Mii ngugc
Dung djch dfm AMP x 5 (Femnentas) gim:
+ 750mM Tris - HCI (pH = 8) + 200mM (NH4)2S04
+ 0,1%(v/v)Tween20 + dNTPs
+ 2mM MgCb
Taq - polymerase (Fermentas) DNA mau
Nudc khir ion
Ting cpng
T h i tich (nl) 1+1+1 1+1+1
5
0,62ni thich hgp vira du 25^1
25^1
Ning df 3,2y.M/\i\
3,2\iMlii\
500UI (1 U/1 nl)
Bdng 3. Cdc chu ky nhift cua phdn ung PCR dung d i xdc djnh gen VT1, VT2 vd eae Cac giai doan cua phan irng
Giai doan tien biin tinh Giai doan biin tinh Giai doan bat cap Giai doan tong hgp Giai doan keo dai
Nhift dp CC) 94 94 50 72 72
Thdi gian (phut) 5 1 1 1 7
S6 chu ky 1 30 1
Ban diu, phdn umg PCR don dugc thyc hifn vdi timg c$p moi rieng bift. Sau dd, phdn umg duge thue hifn vdi sy phdi hgp ding thdi ciia 3 c$p mii. Kit qud thl hifn d bdng 4.
Bang 4. K i t qua thii> nghifm phdn irng PCR dan vd da mii d i phdt hifn mdt sd gen ddc lyc cua VTEC Cac chiing kiem tra
FD523 FD636 C-600
PCR dan m i i VTl
+ +
VT2 + +
eae +
PCR phirc (da mii) VTl
+ +
VT2 + +
eae +
Phan tfeh sdn phim: Cdc sdn phim ciia phdn irng PCR duge tiln hdnh ch^y difn di trfn th^ch agarose 2% trong dung djch 1 x TAE vdi hifu difn thl 50V trong 30 phiit. Dpe kit qud vd chyp anh bdng hf thing Gel Doc 2000. Anh dugc luu giQ- li^i vd dugc tien hdnh xur ly kit qud khi can thilt.
KcM qud cho thiy: Phdn umg PCR phirc eho cac sdn phim rieng bift vd in djnh nhu trong phdn irng vdi timg c^p mii dom. Ngodi ra, phdn img PCR phirc c6n thl hifn mOt so uu dilm khdc:
iici kifm thdi gian, cdng lao d6ng, d$c bift Id tifu hao v^t tu, hda chit eho phdn img cung gidm ddng l<e Do dd, de .xac djnh vi khuin VTEC irong thjt. nen sir dyng phdn ung PCR phirc.
3.2. Ket qua thyc hifn phdn umg PCR vdi cdc chiing dii chiimg
Phuang phdp PCR dCing dl xdc djnh 2 lo^ii gen sdn sinh dOc t i (VTl, VT2) vd ylu ti bdm dfnh intimin (eae) dd dugc thiet l$p vd chuin h6a vdi cdc DNA mlu Id cdc chiing vi khuin dii chirng duong vd dii chimg dm. Cdc chiing E.coli dii chiimg duong (n = 10) (do m^t s6 phdng thf nghifm tham chilu vl vi khuin E.coli cung cip) dd dugc xdc djnh chde chdn cd mang tir 2 3 trong si 3 lo^ii gen (VTl, VT2, eae). Cdc chiing dii chiimg dm (n = 10) gim cd: vi khuin E. coli (C-600, FV847a, FV2289, FV2586, FV2593), Salmonella typhimurium (FD675), P muitocida (PM.VT3), S. suis (HN.25), S. aureus (FD231), C. perfringens (BCD6). Kit qud thyc hifn phdn img PCR phiic dd duge ehuin hda dimg dl xdc djnh 3 gen (VTl, VT2 vd eae) vdi 10 ehiing doi chiimg duong vd 10 chiing dii chiimg dm dugc trinh bdy d bdng 5.
Bdng 5. Kit qud thyc hifn phdn irng PCR da mil vdl cdc chung dii chimg
Ky hifu chiing
Ooi chirng duong
O i i chirng
dm
E. coli FD523 E. coli FD526 E. coli FD528 E. coli FD635 E. coli FD636 E. coli BDL9.1 E. coli BKT29.1 E. coli E4 E. coli E7 E co//E41
Eco//(C-600) (K-12) E. coli FV847a E. coli FV2289 E. coli FV2586 E. coli FV2593
S. typhimurium (FD675) P. muitocida (PM-VT3) S. suis (HN-25) S. aureus (FD231) C. perfringens (BCD6)
Ode tinh v i mOt s i y i u t i gdy bfnh cua vi khuin E. coli
VT1Arr2 VTl VT2 VT1A/T2/eae/Hly VT1/VT2/eae/Hly
VT2 VT2 VT1Arr2 VT1/VT2 VT1/VT2 F4/STa/STb/LT F18/STa/STb/VT2v
F4/STb/LT F18/STa/STbAn-2v
K i t qud phdn i>ng PCR VT1/VT2
VTl VT2 VTIAn'2/eae/Hly VT1An"2/eae/Hly
VT2 VT2 VTIAn'2 VT1/VT2 VT1/VT2
-
Nhu vay, cd thl kit lu|in: phdn ling PCR dd dugc chuin hda trong nghien curu ndy dimg de xdc djnh 3 loai gen ed dp ddc hifu Id 100% khi dugc xem xft thur nghifm trfn cdc chung doi chumg.
KHOA HQC KY THUAT THU Y TAP XIX SO 1 2012
• 1 l - N I'S
Anh 1. Cac san pham cua phan u-ng PCR vdl cdc chung doi ehu'ng duvng vd dm sau qud trinh dif i Ghi chli: M: 100 bp marker, Giing I: FD523 (VTI/VT2), Giing 2: FD526 (VTl), Giing 3: FD528 (VT2),
Giing 4: FD635 (lTl/VT2eaeJ. Giing5: FD636 (VTl/VT2/eae), Giing 6: BDL9.1 (VT2),
Giing 7: BKT29.l(VT2). Giing8: E4 (VTl IT2). Giing 9: E7 (VT1/VT2), Giing 10: E4I (VT1/VT2), Gieng ll, 12. 13, 14, 15: Cdc chung doi chirng dm.
3.3. Ket qua xac djnh dp nh^y vd dp d$c hifu ciia phutmg phap PCR diing de xdc dinh VTEC
Ky thuat PCR hifn dang duge ddnh gia la mpt phuang phdp ed dp nhay vd dp ddc hifu rat cao (Le Thj Tuyit Trinh, 2007). Sd dT co dp nhay va dp ddc hifu cao nhu vdy la do nguyen ly cua PCR sii dyng cdp mii gdn ddc hifu vdo doan DNA dich. Khi thyc hifn phdn umg PCR, ngudi ta nhan thay dp nhay cua phdn ling cung chinh la gidi han phat hifn cua phdn ung. Gidi han nay chinh la ndng dp eiia doan DNA dfch
trong hon hgp phdn urng. Ben canh d6, dp ddc hifu eiia phdn umg PCR la dO ddc hifu cua cae cdp mii sur dyng. Cdp moi phdi dugc lya chpn sao eho cd khd ndng gh6p cdp tdt vdi trinh ty d 2 dau eua doan DNA dfch. Kit qud xdc dinh dp ddc hifu ghi nh^n dugc con cho thdy do^n DNA dfch dugc lya chpn dl khuleh dai cd trinh tu cac nucleotide trong doan gen tuong ddi in djnh va xuat hifn vdi tan sd cao d cac chiing vi khudn cdn nghien curu, nhung lai khdng thdy d cae chiing vi khuin khde.
Bang 6. Kit qud xdc djnh dp nh^y vd dp d$c hifu cua phdn vng PCR tren mdi tru'dng nudi cdy N6ng dp
pha loang CTBO
10-^
10-^
10"^
10-^
10"^
10"^
10-^
10'^
Kit qua FD523(x10°VK/ml)
LB (6,26) + + + + + - - -
m-TSB (6,6) + + + + +
^
BHI (2,375) + + + + + + - - -
FD636(x10%K/ml) LB (5,70)
+ + + + +
= - - -
m-TSB (6,2) + + + + + - -
=
=
BHI (2.508) + + + + + + - -'
Ghi chu: CTBD: Canh triing ban ddu
+: Phdn img duang tinh v&i cd 3 logi gen VTl, VT2 vd eae -: Phdn img dm tinh v&i cd 3 logi gen VTl, VT2 vd eae
Kit qud bdng 6 eho thiy:
v l dp d§e hifu eua phdn img: DNA dugc tach ra tir 3 lo^i m6i trudng nudi ciy diu eho edc phdn img PCR duong tfnh vdi dO d(lc hifu Id
100% vdi ed 3 lo^i gen VTl, VT2 vd eae.
Ve dO nh^y ciia phdn irng: vdi cd 3 lo^i moi trudng, ngudng gidi h^n dudi hay m|it d i cua vi khuin dl tir dd cd thl dimg dl phdt hifn dugc VTEC trong mdi trudng bdng phdn irng PCR Id tuong duong nhau, 2,3 6,6 x lO" vi khudn/ml.
Trong mOt nghien ciru dugc tiln hdnh ndm 2007, tdc gid Le Thj Tuyit Trinh dd tiln hdnh xdc djnh d i nh^y vd dO d$e hifu eiia phdn urng PCR nhdm hodn thifn hf thing PCR phiic dl xdc djnh cdc E.coli gdy tifu chdy. Vdi cdc e$p mii VTl, VT2 vd eae dimg dl xdc djnh nh6m EHEC, tdc gid dd thu dugc kit qud v l dO i\h^y ciia phdn img PCR phirc Id 10* vi khuan/ml. So sdnh giOa 2 kit qud nghien curu, phdn urng PCR cua chimg tdi ed d6 nh^y cao hon do c6 thl phdt hifn dugc s i lugng 2,3 - 6,6 x lO"* vi khuin/ml.
M 3 4 8 9
Anh 2. Cdc sdn phim ciia phdn irng PCR vdi cdc ndng dd pha loSng khdc nhau tip mdi trirdng LB da nudi c i y chung FD523
Ghi chu: M: 100 bp marker, Giing 1: N/c, Giing 2: ndng do pha lodng ICf' (VTl/VT2/eae), Giing 3: ndng ddpha lodng iaUVTl/VT2/eae), Giing 4 ndng dd pha lodng l(fUVTl/VT2/eae), Gieng 5: ndng dd pha lodng iaUVTl/VT2/eae), Giing 6: ndng dd pha lodng I0'\ Giing 7: ndng dp pha lodng KT*. Giing 8: ndngdg pha lodng Iff' Giing 9: ndng dd pha lodng KT*
3.4. Ket qua xdc djnh d$ nh^y vd d i d$c hifu ciia phuorng phdp PCR dung de xdc djnh VTEC trong miu thjt Sfcb
Thi nghifm duge bo trf nhu sau:
Chpn 6 mau thjt (bd, lgn, gd) dd dugc xdc djnh Id am tfnh trong phdn umg PCR dl tiln hdnh gay nhiem vdi cdc chiing vi khuin doi chiimg duong.
25g thjt dd dugc cdt nhd vd cho vdo 225ml mdi trudng m-TSB.
Mdi trudng (m-TSB) dd nudi ciy vi khudn chung FD523 vd FD636 d 37°C/24 gid. Pha lodng thdnh cdc ning dd tir 10"' din 10*
Kit qud dugc trinh bdy d bdng 7.
KHOA HQC KY THUAT THU Y TAP XIX SO 1 - 2012
Bdng 7
Nong dp pha lodng
(m-TSB) CTBO
10-^
10-^
10"^
10-^
10"*
10"^
10"^
10"«
Kit qud xdc djnh dd nh^y vd dd d$c hifu cua phdn d'ng PCR trong miu thjt s^ch Kit qud
FD523(6.6x10^VK/ml) Lgn
+ + +
B6 + + +
Gd + + +
FD636(6,2x10''VK/ml) Lgn
+ + +
Bd + + +
Gd + + +
Kit qud bdng 7 eho thiy:
Ve dp d^e hifu eiia phdn ung: DNA dugc tach ra tir mdi trudng m-TSB cd cdc miu thjt deu cho cdc phdn umg PCR duong tinh vdi dp d^c hifu la 100% vdi ed 3 lo?ii gen VTl, VT2 vd eae.
v l dp nhay eiia phdn ling: Vdi cd 3 lo^i thjt, ngudng gidi han dudi (Lower limit) vl si lugng ciia vi khudn VTEC dl cd thl phdt hifn dugc trong mdi trudng cd cdc mau thjt vd da dugc gay nhilm vdi sd vi khudn dat ndng dp cudi cimg la 6,6 x lO*" vi khudn/ml ddi vdi chiing FD523 va 6,2 x lOSi khudn/ml ddi vdi chung FD636. Hay noi each khac, s6 lugng vi khuan VTEC nhiem trong 25g thjt tuoi phdi dat d mirc 6,2 - 6,6 x 10 vi khudn thi mdi ed thl xdc djnh duge bdng phuang phap PCR nhu da duge chuan hda trong nghien cim nay.
rv.KixLuAN
Kit qua nghien ciiu cho thdy phdn img PCR phiic cho cdc sdn phim rieng bift va on djnh nhu trong phdn ling vdi timg cdp mdi don. Ngodi ra, phan img PCR phirc edn thl hifn mpt sd uu diem khac. Do do, dl xdc djnh vi khuan VTEC trong thjt nen su dung phdn umg PCR phiic.
DNA duge tdch ra tir mdi trudng m-TSB cd cac mdu thjt diu cho cac phdn umg PCR duong tfnh vdi dp dac hieu la 100% vdi cd 3 loai gen VTl,VT2vaeae.
s i lupmg vi khuin VTEC nhiem trong 25g thjt tuoi phdi d?t d miic 6,2 6,6 x 10* vi khuin thi mdi cd thl xdc djnh duge bdng phuong phdp PCR nhu da dugc ehuin hda trong nghien eiiu nay.
Phuang phdp PCR phiic vdi 3 loai e^p mdi (VTl, VT2 vd eae) vd cdc dilu kifn phdn img nhu da duge thilt lap va ehuin hda trong nghien eiiu nay cd thl duge dp dyng df xdc djnh vi khuin VTEC.
TAI up] THAM KHAO
1. Nguyin Binh Minh (2004), Ky thudt PCR da mii xdc dinh cdc loai Escherichia coli gay bfnh, Tgp chi y hgc Viet Nam, si 7, Ting hfi y dugc hpc Vift Nam, tr. 1 - 4.
2. Le Thi Tuyit Trinh (2007), Phdt triln va hoan thifn hf thing PCR da mii xdc djnh tryc tilp cdc Escherichia coli gay tieu chdy tir phdn, Lu§n vSn th?c sy y hpc, D?i hpc Y Ha Nfi.
3. Paton J.C. and A.W.Paton (2002), "Direct detection and characterization of Shiga toxigenic Escherichia coli by multiplex PCR for stxl, stx2, eae, ehxA and saa". Journal of clinical microbiology. Vol. 40, No. 1, p. 271-274.
4. Saiki R.K., Gelfand D.H., Stoffel S., Scharf S.J., Higuchi R., Hom G.T., MuUis K. B. and Eriich H.
A. (1987), "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science, 239, p. 487-491.
