Tgp chi Cong nghe Sinh /ioc 11 (1): 191 -196. 2013
N G H I E N C U U P H A T H I E N SU' C O M A T C U A \ I K H L . A N BACILLIS A\THR4CIS T R O N G D A T B A N G P H U O N G P H A P P C R
Nj;u>cn Phirong .Minh , Dang Van Tien , Ngd Dinh Binh^
' Vien Hda hgc - Mdi Iruang qudn su, Bd Qudc phdng ' Vien Cdng nghe s inh hgc, Vien Hdn tdm Khoa hgc i
TOM T A T
I Cdng nghe Viet Nam
Muc Ueu ciia nghien cuu nay nhSm xay dung phuang phap PCR phat hien nhanh su co mai eua \ i khuan gay benh than Bacdlus anthracis (Ba) trong mdi irudng bang phuang phap PCR. Trong bai bao nay chiing idi da Sli dung phucmg phap PCR de khuech dai gen pagA ma hda cho khang nguyen bao ve PA (proiectne antigen) nam tten plasmid PXO1 la nhan td dan truyen cho cac ddc id gay chei \ a gay phii thung EF, LF cua \ i khuan Ba, nham muc dich phal hien su ed mai cua vi khuan Ba Irong dat. Su dung cac cap moi P.\5 \ a P.A8 de khuech dai doan gen pagA cua vi khuan Ba co kich ihudc 596 bp. cac mau dat su dung irong nghien cinj duoc tao ra bang each gay nhilm bao tu vi khuan Ba d ndng do 10"' - 10"" CFUg va cac mau dat ihu ihap ngoai lu nhien da dugc xac dmh nhiem vi khuan Ba bang phirong phap phan Ioai sinh Iy sinh hoa DNA tir dat diroc Iach chiei true Hep tir dat va su dung kit (EZ-10 Spin Column Soil DN.A Mimi Preps Kit cua hang Bio Basic Inc) de tach chiel DNA true Hep tir dat lam ddi chung Ket qua dat duoc cua nghien cim nay da va\ dimg thanh cong quy trinh PCR phat hien iruc Iicp su ed mai vi khuan than irong dal bang phuang phap PCR. su dung DNA duoc Iach true Uep tir dat vdi dd nhay cao (nong dd vi khuan than trong dat thap khoang lO' CFU g) \.i trong thdi gian ngan khoang 12 gid, Tir ket qua nay chiing tdi cho rang cd the dng dung phirong phap n.iy de phat hien su cd mat cua vi khuan than trong mdi iririmg
Til' khda: Benli Ihan, gen pagA, vi khudn Bacittiis anthracis
Md DAU
Khang nguyen bao ve PA la lhanli phan quan trong trong qua trinh gay ddc cua vi khuiin B anthracis, Phan tir PA hoan chinh cd khdi lutmg 83 kDa, gdm 735 amino acid, kich Ihudc khdng gian 3 chieu la 100 x 50 x 30 A. Khang nguyen bao ve PA do gen pagA ma hoa, gen nay nam Iren plasmid pXO 1(185 kb) (Chauhan el al . 2 0 0 1 , 2002)
Mac du ban than PA khdng gay ddc nhimg nd lai cd kha nang truyen dan cac dpc td vao ben trong te bao vat chu. PA83 cd kha nang ket hap vdi thu the Iren be mat te bao. Dirdi tac dung cua protease ndi bao ho furin, PA83 se bi phan tach thanh 2 phan la PA20 va PA63, de Id ra vi tri hen k6t vdi LF va EF (Mock.
Fouet, 2001; Meselson, 1994) Day !a budc rat nguy hiem ttong qua trinh gay ddc cua PA Cac nghien ciru gim day cho thSy ndu tren phan tir PA83 khdng chira vj tti phan cat nay thi khdng cd kha nang gay ddc Sau do, cac manh PA63 se hen ket vdi nhau de tao ihanh vdng heptam cd 7 nhanh Khi vdng heplam k^t hap vdi nhan td LF se gay chdt va ket hap vdi nhan td EF se gay phii thung cho te bao vat chu Vi va>. khang nguyen bao ve PA duac coi la tac nhan gian tiep gay ddc cho \k bao vai chu lam tang hieu qua gay ddc cua
nhan id gay chet va phu ihung (Mock. Kouci. 2001, Meselson. 1994)
Tren the gidi hien nay cd rat nhicu ioai kil phal hien Ba khac nhau, cd the dua ircn nguyen ly mien dich hoac phuang phap smh hoc phan lu Tuy nhicn.
tai Viet Nam chua cd loai kit nao duac su dung phd bien Tu nam 2003 nhdm nghien cuu cua chung tui da cd nhumg cdng bd dau tien ve \iec xay dung cac phuong phap phal hien benh than thdng qua cac dJc tinh sinh Iy. smh hda va thdng qua \icc phal hicn gen ma hda khang nguyen bao ve PA cua \i khuan B anthracis (Ng^d Dinh Binh el at. 201)3. 2004| "luy nhien cac phuong phap nay chua hieu qua \i tdn nhieu thai gian \ a hda chat cho qua tnnh xac dinh cac dac tinh smh ly, smh hda
VAT LIEU PliUONG PH.AP
\ ' a t lieu
Ba mau dat duac lay nhiem \i khuan than dn Hpc vien Quan > ^^ung cap
Ba mau dat nhiem vi khuan than do plionu I)i iruven \ i sinh \at - Vien Cdng nglic smh IH-L IIIIIL' i..ip
I'll
Nguyen Phupng Minh eiai Ddi chimg dirong la vi khuan B. anthracis
VCM1167
Cap mdi de khuech dai gen pagA ciia vi khuan '. anthracis cd trinh tu n h u sau.
Ten m6i PA5 PAS
Trinh tip n u c l e o t i d e 5'-TCGTAACACTAACGAAGTCG-3' 5 -GACGTAGAAGGATATACGGT-3
Gen pagA
K i c h thu'o'C (bp) 596
Phu'tmg p h a p nghien ciiu
Phirfftigphap chudn bi DNA vi sinh vgt tir dat (cdi hien 1 phan phuotig phdp cua Phgm Kieu Thuy el.
ai, 2008) de phuc vtt viec phiil hien gen pagA ndm Iren plasmid pOXI
Do gen pagA nam tren plasmid nen cd the sir dung phuong phap tach chiet DNA plasmid bang dung dich I. II, III d phan dudi de chuan bi DNA dung Iam sai khudn khuech dai gen. I g dat duac lac trong 10 ml nudc cat vd triing trong vdng 30 phiit Ly lam chuyen dich vao mdi trudng dinh dudng, lac a 200 vdng/phiit trong thdi gian 5 - 7 gid. Tiep theo djch nudi cay duac chuyen vao dng eppendorf, ly lam 6000 v/p, trong 10 phiil, thu can. Bd sung vao mdi dng 150 pi dung dich cac loai Sol 1 , Sol 2 , Sol 3 de pha te bao Sau dd, ly tam 10.000 vdng/ phiit, Irong 10 phiil a 4"C, Thu dich ndi Bd sung vao mdi dng 1 the tich cdn tuyet ddi den 1,5 ml, u d -20''C trong 3 gid. Sau dd, ly tam lanh 10.000 vdng/phut trong 10 phiit a 4"C, bd dich de khd tu nhien. Hoa lan DNA plasmid trong dng eppendorf da dupc lam khd bang 45 pi Rnase de loai bo RNA Irong vdng 30 phiit - I gid a 37"C DNA dupc bao quan o -20"C va diing de chay PCR phat hien vi khuan B, anihracis bang cap mdi PA5 va PAS
Dung dich Soi I Tris - HCI IM (pH8) 2,0 ml;
EDTA 0.5M (pH8) 1.6 ml:
Glucose 0,72 g; H 2 0 khu ion KVi 80 ml;
Dung dich Sol II NaOH 3M IOO pi. SDS 10%
1 5 0 p I ; H 2 O k h u i o n 1250 pi
Dung dich Sol III CH3C0OK 3\1 23.52 g;
C H 3 C O 6 H 9.2 ml
Tach DNA tdng sd ciia \'SV trong daf bang kit (EZ-10 Spin Column Soil DNA Mimi Preps Kit cua hang Bio Ba%\c Inc).
Phuo-ng phtip chiet DNA vi khudn
Vl khuan dupc nudi lac qua dem tren mdi Huong MPB a37"C vdi tdc dp lac 200 vdng/phiit. Te hao lir 1.5 ml dich nudi duoc thu Iai bang phuong
phap Ii tam vdi tdc dp 6000 vdng/phiit trong I phuL Tk bao dupc rua 3 lan bang IOO pi dHiO va duoc hda Iai trong 70 fil dHiO, DNA cua vi khuan duoc giai phdng ra ngoai dung dich bang each dun sdi vi khuan d IOO"C trong 10 phut va dupc Ihu Iai bang each Ii tam nhanh DNA duac bao quan d - 20''C.
P h u t m g p h a p P C R
Hdn hop phan iing PCR cd the tich 20 pi bao gdm: Taq Buffer 2 pi, dNTPs (10 mM) 2 pi, MgCb ( l O u i M ) 4.SpI, P A 5 ( 1 0 p m o I ) I pi, PAS (10 pmol) 1 (il, Taq polymerase 5U 0,5 pi, DNA khudn 1 pi, dHiO 7,7 pi, Cac hda chat su dung trong phan ung cua hang Fermentas, Cap mdi PAS va PA8 duac thiet ke va duac tdng hap bdi hang san xuat IDT Phan ling PCR duac thuc hien d dieu kien 94''C/3';
30 chu ky, mdi chu ky gdm cac budc 94''C/30", 5 5 " C / r , 7 2 " C / r ; 7 2 ^ / 1 0 ' ; san phSm PCR duoc dien di tten gel Agarose 1% trang dem TAE va chup bang may chup gel Biorad UV2000.
KET QUA THAO LUAN
Xay dtrng p h u o n g p h a p PCR p h a t hien vi khuan Ba trong d a t
Qua thdi gian nghien cuu phat hien vi khuan Ba trong dat bang phuang phap phan lap chiing Idi nhan Ihay day la phuang phap mang lai hieu qua khdng do thdi gian thuc hien keo diu, tdn kem nhieu hda chat va van phai sir dung phuang phap PCR de khang dmh chinh xac cd phai la vi khuan Ba hay khdng Vi vay cau hdi dat ra la: tai sao khdng nghien cuu quy trinh khac hieu qua han va thdi gian thuc hien ngan han'' Tir dd, chung tdi tien hanh hoan thien phuang phap thuc hien bang each thuc hien tach chiet true Iiep DNA tdng sd cua vi khuan trong dat de thyc hien phan img PCR nham khac phuc nhihig nhugc diem tten
DNA long sd ciia vi khu^n ttong dit dupc thtrc hien tach chiet theo quy trinh da dupc neu ttong phan \at lieu phuong phap, Trong d i t cd nhilu loai
Tap chi Cong nghe Sinh hgc U( I): 191-196,2013
Vl khuan khac nhau, vi vay DNA thu duac la cua va lrung tam hoat ddng cua enzyme laq poKiuerase.
nhieu loai vi khuan khac nhau. vi vay chiing tdi tidn hanh khao a cac ndng dp mdi va taq polymerase khac nhau de khao sat diem tdi uu cho phan img PCR. Ket qua duoc the hien qua hinh 2.
Tir ket qua Uen nhan thay d ndng dd mdi 15 pmol va 20 pmol ket hap vdi 0.5 pi taq polymerase 5U xuat hien bang cd kich thudc 596 bp diing nhu tinh toan ly thuyet, cdn d cac ndng khac thi khdng Dieu dd chung td ndng dp taq polymerase va mdi tdi uu cho phan ting PCR khuech dai gen pagA cua \i khuan Ba trong dat la 15 pmol mdi va 0,5 pi taq polyiuerase,
Sau khi xac dinh dupc ndng dp ihich hop ve mdi va taq polymerase chung tdi khao sat gidi ban mat dd vi khuan Ba trong mau dat bang each iao ra nhumg mau dat cd mat dp bao tu tit lO' den 10" CFU/g dat.
Sau dd. tien hanh tach chiet DN.A tdng sd cua vi sinh vat trong dat va thyc hien phan img PCR theo dieu kien da dupc tdi uu tren.
Hinh 1, San pham tach chiel DNA long s6 trong dal
Ben canh dd, DNA dupc tach tryc tiep tit dat cd chua cac acid huu co va kim Ioai nang lam irc che qua trinh phiin img PCR do chung ket hop vdi DNA
Hinh 2, Khao sat cac di^u kien phan ung PCR khuech dai gen PagA 1,2 khao sat n6ng do m6i 15 va 20 pmol va 0,5 ^ll taq polymerase 5U, 3 Ihang DNA chuSn, 4 khao sSt nfing dp moi d 10 pmol va 0,5 pi taq polymerase 5U, 5,6 khao sal ndng dp mdi a 15 va 20 pmol va 0,2 y\ taq polymerase 5U
Hinh 3. San pham PCR tu- DNA long so cua vi San pham PCR cua cac DNA lach chi§l l u 6k cb nong dp baotu' l O ' d f i n 10', 4 DC (+) DNA cua chung VCW1167, 5 Thang chuan DNA
Kat qua thu dupc san phim PCR tu DNA tach d ca 3 mau dal cd ndng dp bao tu vi khuan ihan tir 10 den ID' diu xuil hien bang cd kich thudc khoang 596 bp tuang duong vdi kich thudc ciia ddi chimg duong Dilu dd chimg lo hieu qua ciia phuang phap rai cao 100% cac mau diu duang tinh va cd the phat hien VI khuan Ba trong dat d ndng thap lO' CFU/g,
Sau khi tdi uu dupc dieu kien phan ung PCR cho phat hien gen pagA chiing tdi tien hanh su dyng quy trinh nay de phat hien cac mau dat thu Ihap ngoai tl/ nhien da duac xac dinh la cd vi khuan
Ba bang phirang phap su dyng cac phan img iinh ly sinh hda. do phdng di iruycn \i smh \icn Cdng nghe sinh hoc cung cap, Kel qua ihu dupc 100%
cac mau dd deu cho ket qua duong linh, \di tdng ihdi gian thuc hien theo phuang phiip nay da duoc riit ngin xudng 12 h. Nhu vay chimg idi dii xay dimg dupc phuong phap PCR dc phat hicn su cd mat cua \ i khuan Ba trong dai \di thdi gian time hicn ngin htm phuang phap sinh 1\ sinh hda ca ban da diing irirdc day cua phimg Di iruycn Vi sinh \at - Vien Cdng nghe smh hpc.
I';3
Nguyin Phuang Minh et at
Hinh 4. Kgl qua phal hien vi khuan Ba trong dat bang phuong phap tach DNA VI khuan trifc tiep tu'dSt 1,2,4 cac mau flat nhiem Ba da phal hien bang phucmg phap phan lap (DDB3, DDB8, DDB22), 3 thang DNA chuan, 5 DC (*) m^u dSt d u o c gay n h i i m uai chiing vi khuSn Ba VCM 1167, 6 DC (-) m§u dSt khong co vi khuan Ba
Phat hien vi khuan Bacillus anthracis su dung DNA tong sd tach Irong dat bang kit (EZ-10 Spin Column Soil DNA .Mimi Preps Kit ciia hang Bio Sflsic Inc).
Hinh 5. Kel qua dien di san phan- b^ng kll tmh sach EZ-10.
lach chiel DNA trong dac
Dd so sanh dp chinh xac cua phirong phap PCR vdi DNA tdng sd tach tryc tiep tii dat, chimg tdi sir dyng bd kit tach chilt DNA ciia hang Bio Basic Inc lam ddi chiing. Day la bd sinh pham diing de tach chilt DNA tir dit vdi nhung DNA cd kich thudc >
20kb, DNA cd kich thudc nhd se bj rua trdi qua cdt.
Gen pagA nam tren plasmid pOXI cd 185 kb vi vay se duac giiJ Iai trong qua ttinh tach chiet.
Sau khi tach chiet DNA thanh cdng chiing tdi tien hanh xac djnh nong dp vi khuan ttong dat cd the phat hien dupc hay ndi each khac la khao sat gidi ban mat dp vi khuan trong mau cd the phat hien dupc bang phuang phap PCR vdi DNA dupc tach bang Kit cua hang Bio Basic Inc
Hinh 6. San phSm PCR khao sat nong do ciia vi hhuan trong mSu dat (DNA tach bang kit E Z - 1 0 ) 1 Thang chuan DNA; 2-4: San pham PCR t u DNA ciia cac m l u akl co mai dd 10^ den 10^ CFU/g
Ket qua thu duac sau khi thyc hien phan img PCR nhan thay chi xuat hien bang cd kich thudc -596 bp d san pham PCR cd ndng dp vi khuan 10 va IO** dieu dd chimg td gidi han vi khuan cd the phat hien khi sii: dung Kit EZ - 10 de tach triet DNA la 10' CFU/g.
Hinh 7. San pham PCR phat hien gen pagA cua DNA tach l u cac m i u dSl 1-3 san pham PCR vdi DNA tach bSng phuang phap long so (DDB 3, ODB 8, DDB 22). 4-6: san pham PCR t u DNA tach bang kit E2-10 {DOB 3, ODB 8, DDB 22) 7 DC (+] D N A c u a c h u n g VCM1167, 8 Ihang c h u i n DNA
Sau khi khao sal Irong dat cd the phat 1
!idi han mat dp vi khuan Ba ien diroc, chiing tdi tien hanh
thirc hien phan img PCR khulch dai gen pagA cua vi
khuan Ba trong cac mau dit da phat hien cd vi khuin
Tap chi Cong nghe Sinh hoc U(l): 191-196,2013 Ba bang 2 phuang phap tren vdi dieu kien phan img da dupc tdi uu tuang ly nhu phuang phap tach chiet DNA tdng sd cua vi khuan trong dat.
Ket qua thu dupc cac mau tren deu cho phan ung duong tinh (chi xuat hien bang DNA cd kich thirdc -596 bp tuang duang vdi ddi chiing duang chung VCMl 167) vdi tdng thdi gian thyc hien khoang 6 h, Dieu dd chimg id bang phuong phap nay Ihdi gian thyc hien da dupc riit ngan xudng 6 h so vdi phuang phap tach DNA tdng sd
KET LUAN
Trudc day, de phat hien vi khuan Ba bang phuang phap phan lap can khoang 40 - 48 gid, Tuy nhien vdi phuang phap PCR su dyng DNA tach chiet trirc tiep tir vi khuan trong dat chimg tdi da giam di kha nhieu thdi gian de phat hien (chi cdn 12 h) ddng thdi khdng can tdn kem qua nhieu ve kinh phi cung nhu nhan lyc Iam viec, dac biet phuang phap nay cd the phat hien duoc vi khuan Ba trong dit d mat dp thip la 10' CFU/g.
Ben canh dd, phuang phap PCR su dyng DNA lach chilt bing kit EZ - 10 cd uu diem la da giam tdi da dupc thai gian phat hien chi cdn Iai 6 h Tuy nhien, phuong phap nay cd nhupc dlem la tdn kem chi phi cho viec mua kit Iach chilt DNA va khdng the phat hien duoc khi mat dd vi khuan trong mau < IO' Ldi cam o'n: Cdng Irinh cd su hd Ira ciia Phdng thi nghiem li'ong diem Cong nghe Gen, Vien Cdng nghe smh hoc, Vien Him tdm Khoa hoc vd Cdng nghe Viet Nam
TAI LIEU THAM KHAO
Andersen GL. Simchok JM. Wilson KH (1996) Identification of a region of genetic variability among
Bacillus anthracis strain and relaled species. J Bucierial 178-377-384.
Bell CA, Uhl JR. Hadfield TL. David JC. Mever RF.
Smith TF, Cockenll 111 FR |2002) Deiection of'Bacillus anihracis DNA by LisihtCvcler PCR J Clin .Microbiol 40 . (8): 2897-2902
Chauhan V, Bhatnagar R (2002) Identiflcauon of amino acid residues of anthrax protecUve antigen invohed in binding with lethal facior Infect Immiin 70(8) 44"''-1484 Chauhan V, Singh A Waheed AM. Singh S. Bhatnagar R (2001), Constitutive expression of protective antigen gene of Bacillus anthracis in £ co//, Biochem Biopin Research Commun 283(2) 308-315
Makino SL Cheun HI, Wataral M. Uchida I. Takeshi K (2001) Detection of anthrax spores from the air by real- Ume PCR. Leu App Microbiol 33 237-240 Mock M, Fouet A (2001) Anthrax. Annu Rev Mcrohiol 55 647-671
Meselson M, Guillemm J. Jones MH (1994) The Sverdlovsk anthrax outbreak of 1979 Since 266(5188]
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Ngo Dinh Binh, Nguven Xuan Canh. Nguyen Thi Anh Nguyei, Hoang Ngoc Hien, Ngnyen Thai SOTI. Le Thu Hdng (2004) Phan lap va phan loai vi khuan Baciltu'^
anlhiucis tir benh nhan mac benh than o Vicl Nam Nhuny vdn di' nghien nm co- ban Irimg khoa lioi \ir song, dinh hirirng Sinh - Y hoc
OkinakaRT. Cloud K. IlamptOTi O. HotTniusUT AR. Ihil KK, ct al (1999) Sequence and organi7iition of pXOl, llie large Bacillus anthracis and ils possible use in diagnosis EE,MS Immunol Med Muiohiol 15 233-14 Pham Ki^u Thuy, Pham Mmh Tuan, Dmh Duy Khang, Ngd Dinh Binh (2008) Bieu hien va tinh sach khang nguyen bao ve PA lai td hop cua M khuan B anthracis Tap clii Cong nglii- Smh hoc 6(4A) 743-750 Weikos SL, Lowe JR, Eden-McCutchan F, Vodkin M.
Leppla SH. Schmidt JJ (1988) Sequence and analysis ol DNA encoding protective antigen of Bin dins cmlhrcicts Gene 69 (2). 2h-300-
Nguyin Phupng Minh et ai
D E T E C T I O N O F BACILLUS ANTHRACIS IN S O I L U S I N G P C R M E T H O D Nguyen Phuong Minh'"*, Dang Van Tien^, Ngo Dinh Binh"
'Centre for Technology Environmental Treament, Ministiy of Defence ' Institute of Biotechnology, Vietnam Academy of Science and Technology
SUMMARY
The aim of this study was to develop a polymerase chain reaction (PCR) procedure for detection of Bacillus anthracis in environment hi this study we used PCR and primer pairs PA5 and PAS lo amplify a DNA fragment of 596 bps located in pagA gene encoding protein protective antigen (PA). This antigen is a transmission for the lethal factor toxin and edema facior toxin of S anthracis bactena. The soil samples used in this study was created by infecting B anihracis spores al a concenlraUon of 10"' - I C CFU/g and soil samples contaiminaled with B anihracis proved by biochemical and physiological classification method DNA templates for PCR reacUon were extracted by 2 methods. The fist DNA extraction direct from the soil and the second DNA extracUon direct by EZ-10 Spin Column Soil DNA Mimi Preps Kit by Bio Basic hic. Using DNA template exlracled directly from Ihe soil had gave high sensiiiviry(the concentration of B anthracis about lO' CFU/g) and reduced lime course to 12 hours for detection. The PCR meihod can be applied to detect anthrax in environment
Keywords: Anilvax disease B anihracis. pagA gene
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