VIEN SOT RET K''''? SINH TRUNG CON TRUNGTRUNG UONG BAO CAO KET QUA DE TAI NGHIEN CN CAP VIEN TEN DE TAI AP DUNG KY THUAT PHAN LING CHU ©I POLYMERASE (PCR RFLP) DE PHAN BIET GIUN DUA NG UCI I VA GIUN DUA[.]
VIEN SOT RET- K'? SINH TRUNG - CON TRUNGTRUNG UONG BAO CAO KET QUA DE TAI NGHIEN CN CAP VIEN TEN DE TAI: AP DUNG KY THUAT PHAN LING CHU ©I POLYMERASE (PCR- RFLP) DE PHAN BIET GIUN DUA NG UCI I VA GIUN DUA LO N Chis nhi6m dé tai: Co quan chin tri dé Hi: Th.s Hoang V6n Tan Vi(n Sot rét- Ky sinh triing- Con triing Trung uong Ha Noi, nfim 2007 CN S6T RET- KG SINH TRUNG - CAN TRUNGTRUNG UONG BAO CAO KET QUA DE TAI NGHIEN CN CAP VIEN AP DUNG KY THUAT PHAN LING CHUOI POLYMERASE (PCR-RFLP) DE PHAN BIET GIUN DUA NGLf©I VA GIUN DUA LCJN Chii nhi(m dt: tai: Co quan chu tri dé téi: Th.s Hoitng Viln T8n Vi(n S8t rét- KJ sinh triing- C8n triing Trung uong Vi(n S8t rét- Ky sinh ti ing- Con triing Trung uong Th6i gian thuc hi(n d8 tilt: Tit théng n6m 2006 ddn th:ing 12 nam 2007 H8 N0 - BAO CAO KET QUA DE TAI NGHIEN CLIU CAP VI E) N Tén dé Hi: Ap dung ky thuat phan mug chuoi polymerase (PCR-RFLP) de phan bi(t giun dfia ngubi va giun dfia ton T8n Chis nhi in dc: tai: Th.s.Hoéng Viln Co quan chin tri dé tai: Vi(n Sit rét- Ky sinh triing- Cm triing Trung uong Co quan qu:in IQ de tai: Vi(n Sit rét- Ky sinh triing- C6n trilng Trung uong Danh s:ich nhiing nguéi thuc hi(n chinh: Th.s Hoiing Viln T6n Vi(n S6t rét-KST- CTTU Th.s.Nguy8n Thi Huong Binh Vi(n S6t rét-KST- CTTU PGS.TS.Nguyen Diic Manh Vi(n t rét —KSTCTTU Th.s Le Diic Dao Vi n t rét-KST- CTTU Th.s Ha Viét Vién Vi(n Sit rét-KST- CTTU Théi gian thuc hien:Tit thiing nam 2006 den th:ing 12 n:inn 2007 CAC CHU VIET TAT PCR-RFLP: Phén King chuoi Pholymerase ph6n tich tinh da hinh dfi dai doan ph cat gi‹ii han KST : Ky sinh tritng ‘ SL : SP luong ITS-1 : Vung phién m5 ITS-2 : Vung phién ma ADN : axit dezoxyribonucleic dNTP, : dezoxy nucleotide triphophate dATP : dezoxy adenosine triphophate dGTP : dezoxy guanidine triphophate dCTP : dezoxy cytosine triphophate dTTP : dezoxy thymidine triphophate Taq - Polymerase: enzym polymerase cua Themophilus Buffer : Dung dich dim Primer: Mfii TE: (Tris-base + EDTA) EB: Extraction buffer PBS : phophate buffered saline (D(m ph6t phat) bp : base pair ( Dfii bazo nito ) pl : microlit Marker: Thang trpng liipng phM tu WHO, : To chiic Y te thé gicii Dé tiii duoc hoén thénh tai Khoa Sinh hoc Phtln tu Vi(n S6t rét - KQ sinh trilng — Cfin trilng Trung Uong Hoan dé tai toi da nh duoc su giiip dfi nhi(t tinh ciia Th.s Nguyén Thi Huong Binh, su giiip dfi chu d:to cua GS.TS Nguyen Diic Manh, su cong t:ic nh)p nhang ciia Th.s Ha Viet Vien va c:ic anh chi em Khoa Chilng tot vo ciing cam on su tao di‹:u ki(n thuan loi ciia Lanh dao Vién, dac bi t la su giup dfi va tao dieu ki n ciia Khoa Sinh hoc Ph6n tit, Phong Qu 1y Khoa hoc Vien, Phong Ke hoach Tong hpp va Phfing Ké Toan tai chinh, cung ciic phong chiic nding co lie‘n quan da giiip chiing tfii hoii n thénh de tai diing tién dfi Tfii xin trtin c:inn on moi su giiip qiii b:iu ! Ha N i, 20 th:ing nam 2007 MUC LUC Tran8 PHAN A.TOM TAT KET QUA NPI BAT CUA DE TAI 2.1 Nghien cilu nuoc .4 2.2 Nghi6n ciiu nuoc ngoai .5 T1-HJ GIAN, ! DIEM, DU TUDNG VA PI-I UDNG PI-IAP NG I-H EN C’UU i.i.wai gian nghién emu .8 3.2.Di|a diem nghie‘n erm 3.3 r›oi twong nghién erm 3.4 Phuon g ph:ip nghi6n ciiu 3.4 I Twist ké nghién cim 3.4.3 .C:ic ky thuAt sit dung nghie’n c6u 3.4.3.1 Thu th8p mAu va béo qu:in mtl u 3.4.3.2 PhAn bi I giun dfia nguoi va giun dfia ton bang dltu hi(u hinh th:ii 3.4.3.3 PhAn bi(t giun diia nguoi va giun dua lpn bang ky thullt PG R UT º3 US 12 4.1 Minh liinh nhie’m giun tai diem nghién ciiu 12 4.2 Pht1n biét giun diia nguor va giun diia lori .13 4.2.1 Ph6n but hinh the giun diia nguéti va giun diia lpn 13 4.2.2.Phan biet giun diia ngiiéii va giun diia ton big ky thu9t PCR- RF'LP 16 4.2.2 5:in ph4m PCR ciia giun diia nguéi vé giun dua ton chua cat enzym Haelil 16 2.2.2.Siin pliam PCR ciia giun dfia ngiicri va giun diia ton da cat enzym HaeIH 17 DAN LUAN 18 Kich thucrc giun 18 5.2.I—birth anh rig cfia ascaris lumbricoides vé ascaris sunni 18 5.3.Phiin biet giun dfia nguéri va giun diia ton b:ing ky thu t PCR .18 KET LUAN VG OE NGH ! 20 TAI LIEU THAM KH .21 PHAN A.TOM TAT KET QUA NOI BAT CUA DE TAI QUA CUA OF TAI 1.1.Dong gop miii cua dé tiii: Da bii sung cho phuong ph:ip d]nh loai m8t phuong ph:ip chlln doan mtii (Phuong ph:ip ch±n doan bang ky thult PCR) 1.2 Ket qfia cu thé ciia dé tiii: Chiing Hi da tién hanh ph8n tich 119 m4u giun diia, dfi c‹i 68 mAu giun diia nguéri [Ascaris lumbricoides) va 51 mAu giun dua ton ciia hai xa Phuong Tmng, huye’n Thanh Oai va xa Phung ChAu, huy(n Chuong My tinh Ha TAy, bring phan King chuoi polymerase (PER - RFLP) Ap dung ky thu(t PCR de nhdn viing phie’n ma thii nh:it (ITS- I) o giun diia, sau cat s:in ph±m PCR bring enzym HaeW vt tri 358 dfii vdi giun d fia nguéri cho b rig, v6i kich thuoc c:ie b:ing lan luot la 610 bp va 370 bp Enzym IfneI I I c:it san phAm PCR cua giun dfia ton tai hai v] tri 131 va 358 cho bring, v6i kich thuéic ctic bring 1tin luot la 610 bp, 230 bp va 140 bp Dna vao sG lvong bang cat va kich thu6c ciia c:ie bring, ta co thé ph8n bi(t dune giun dfia nguéri, giun dfia ton Tuy nhién nghién erm chiing Hi ph:it hi(n dune 14,7'7o giun dfia ngiiiii co bring, dfi co mt5t bfing chung cho cé giun dua nguiti va giun dfia lori (kich thu6c 610 bp) va mGt bring cua giun dna nguéi (kich thiidc 370 bp), hai bring lai co kich thu6c triing v6i kich thu‹ic hat bring cua giun dfia Ion (230 bp va 140 bp) 1.3 Hi(u qua trio the: Ning cao trinh d0 thuc hiinh ky thuttt PCR cho cite ciin b0 nghién ciiu 1.4 Hiéu qu:i hinh té vii xd htd Ap dung ph:in ting chufii polymerase (PCR - RFLP) phdn bi(t giun dfia nguéri va giun dua lori da kh:ie phuc dupe nhfmg kho khiin ma phtiong phap dmh loai bling hinh th:ii gap phai M:it kh:ic thong qua ky thu(t t ta co the xtic d]nh dupe kha n:ing nhiém chéo giun dua ton tren nguéii va nguoc lai SA AP DUNG VAO THUC N sinh hoc phdn tit ve ky sinh triing duérng ruot Ung dung thuc tién tai c:ie labo vi(c chlln do:in phttn bi t giun dfia nguéii va giun dua ton DANH GIA DC TAI VIII DE CUONG NGHIEN CIJIJ DA DUtJC DUVET a Dt: tai da thqc hi(n diing ti6n ( tit th:ing 5/ 2006 -12/ 2007) b Da thuc hi(n thiinh cfing phdn bi(t giun dua nguiii va giun dfia ton bring ky thuitt PCll c tai da co bao cao ti6n d0 va bao cao tong két diing théi han d D:inh gia ve su dung kinh phi Dé tai da sir dung kinh phi diing muc dich chuye’n va diing nguyen tac tai chinh Tfing so kinh phi dupe duy(t 46.000.000 ding kinh phi dii su dung 46.000.000 déng 4.DC NGHI : Clin tién hiuih diéu tra tinh hinh nhie‘in che’o giun dfia ngiréii va giun dfia ton trén mot quy mo mo rong 16n hon va :ip dung c:ie ky thu:It sinh hpc phtln tir dé dinh loai thiinh ph4n loai dé d:inh gia dich té hoc ph±n ti'r ciia vi(c nhiém giun diia Ascaris c8ng ding PHAN B N OI DUNG BAO CAO CHI TIET KET QUA NGHIEN CN Bénh giun dfia la mot bénh xa hfii Bt:nh nhiém ci hdu h6t moi lira tuoi, moi gi6i, tit n6ng th6n, thiinh thi den céc viing nili cao T:ic hai bénh chit yéu la chiém chat bo du rig tir thiic tin ciia v6t chu, lam ch6m l6n, chilm ph:it trién tri tu(, benh co th6 gdy tit vong giun chui 6ng in t, tac ru t giun v v Nufic ta la nuoc nong nghi(p, nam vilng nhi(t d‹ii gio miia co dint ki(n h8u, nhi(t dfi, am dfi va tho nhuting rdt thich hpp cho céc bénh ky sinh triing du rig ruo( t ph:it trii i, dac bi(t la b6nh giun truyén qua dlt rdt bién va nhiém o hau het moi lira tuoi, moi gi6i nhung cao hon o lira tufii tré em Vi v y viec phong va chong bé nh giun during ru t la mfit vi(c lém rlt cdn thiet va da tit l6u la noi mong moi ciia nhieu nguéi Trucic tinh hmh dfi, nhiéu nha khoa hoc da di s6u nghie‘n ciiu d é tim c:ic bi(n ph:ip phong chong &nh thich hpp nham lam giém ty I( va cuéng dfi nhiém giun nhdn d , d3ac bi(t la lira tufii hoc sinh C:ic nghién ciiu vé giun truyen qua ddt trufic ddy chin yéu nghién ciiu ve bénh hoc, di6u tra va diéu tri &nh, song ddn cu song t8p trung, diéu ki(n ve ’i truéng sfing quanh ta b) fi nhiém triing giun r8t tr6m (Vo Vi‹:t Him va cdng t:ic via:n, l960; DC Duong Th:ii, Nguyén Phan Long, 1973; Hoiing Thi Kim va cfing t:ic vien, 1987 )[2,3,41 • y ci mien Bac chi sau m6t théi gian ng¾ thiing sau di6u tri co t6i 90Wo so ngu i bi t:ii nhiém giun dua ( vé CS (1976) [5] Trong nhfmg nam gttn d6y, tinh hinh c:ic bénh giun truy8n qua ddt ci nuoc ta ' chua duoc c:ii thi(n d:ing ke Theo diéu tra ciia Vi(n S6t rét Ky sinh triingCfin trung trung nong (KST-CT.TU)1998, cho thdy ty lé nhi6m b6nh giun truyén qua ddt vén cao o ca mién d mien Bac ty 1( nhiém giun dfia tit 50-959c , giun toc tit 58-899o , giun moc tu 3-679o mien Trung ty le nhiém giun dfia tit 12,5—70,§9o, giun toe tir 12,7- 479o, giun moc tii 36- 69Wo 1Tii‘en Nam ty Ie nhiém giun dfia tit 10- 60'7c , giun toe tir 0,5-1,79o, giun moc tit 47- 689o [1] Mat kh:ic, ci nuoc ta giun dfia ngu i va giun dfia ton lufin song song téin tai De gop phdn vao cfing tae phong chfing bénh co hieu qila cao, viec ph8n bi(t hai loai giun dua noi trén co m0t ’ silc quan trong cfing I:ic phong nnghhiiaa hit chong bénh tai cfing dong Vé mat hinh th:ii, kich cfi triing giun va giun dfia truéng tit ngu i va tir v I chu ton la tuong tu nhau, chu k sing ciia hai loai giun giong nhau, v6y kho co thé ph6n biet duoc bang hinh the gifia hai loai giun nfii trén De giai quyét vdn de nay, vi(c tip dung ky thu t ph6n tich tinh da hinh dfi dai doan phan cat gi6i han tit san pham PCR de phan bi(t giun dfia tit nguéi va tir v6t chit lpn la rdt c6n thiet Do v y chiing toi tién hanh d‹: tai: “Ap dung ky thuat phén ring chuoi polymerase (PCR- RFLP) dé phan bi(t giun dua nguéi va giun diia lpn ” Muc dich dé tiii: Ap dung ky thuat phén ting chuoi pholymerase (PCft-RFLP) d8 ph6n bi(t giun dfia nguéi va giun dfia Ion TONG QUAN TAI LI E) U 2.1.Nghién elm nu‹ic Dinh loai dna vao c:ic ddu hi(u hinh th:ii duoc :ip dung tit xa xua va r6t bién cr nhiéu nucic trén the gi6i Phuong ph:ip da dong gop to 1‹in vao vi(c xac dmh thénh phtih loai nghien ciiu noi chung va nghién ciiu c:ic benh k sinh triing nfii rieng, phuong ph:ip dna vao d6u hi(u hinh thai viia kinh té, viia don gi:in, lai nhanh va co thé ap dung tai thuc dia Tuy nhien, phuong phiip cling b6c 16 nhfmg han ché nh:It dinh nhfmg truéiiig hpp m6u vilt khfing dii i hinh, m6u v6t khfing nguyen ven, cac mdu vat co miic dfi tuong dong cao v.v .thi viéc x:ic dinh phan loai cling gap phai nhfmg kho kh nh6t d]nh Do Duong Th:ii,(1971) nghién chin vé hinh the triing, au tiring va giun truéng thdy rang hi(n v8n khfi kh , chua ph6n bi(t duoc gifia giun diia ngufii va giun dfia Ion, vdy chua giup duoc cho dich te hoc mGt c:ich co hi(u qua d nuoc ta, giun dfia ngu i va giun dfia Ion r6t phfi bién va song song tin tai mfit v6n dé Vi(c x:ic dinh hai loai giun la dGng nh6t hay t:ich réi nghien ciiu rht ciin thiét, viia co tinh ting dung phln loai, vita King dung dich te, bénh hoc[1] Theo nghién ciiu cua DC Duong Th:ii (1973), hinh thé bén ngoai ciia triing, du triing giun dua va giun diia truéing thiinh duoc tae gié mo ta khong co gi dac biet so v6i tai lix:u co dién Theo t:ic gié, m6u sac cua giun truéing co sq thay doi theo timg cc thé va timg mdu giun Thir6ng giun dfia co m6u sac thay d6i tir m6u tr¼g sfra d$n in:tu hfing nhat, m6u sac co lién quan d6n siic dé kh:Ing ciia giun T:ic giii cho bic:t, giun co m6u hfing nhat thuéing co khé uang scing lhu hon moi truéng béo t8n gfim nucic sinh ly vé glucoza, m6u sac giun cling kh0ng ’ i cho c0ng t:ic dinh loai[4] Dung kéo nhfi, sac cat init phdn ciia cc the giun ci b6t ky v] tri nao, mau cat nho co kich thuoc (10—20 mg) Dna md u v t vao tube eppendorf 1,5m1, mo nap tube dé cfin bay hcii hét, sau dfi cho them 200pl dung dJch tach (EB) va nghién bang chay chuyén dung den m6u v t o d:Eng hon dich té bao U c:ic tube eppendorf co chiia md u cr 650 C 30 phfit 4.Them vao moi tube 30 pl Kac 8M de kit tiia philc hpp protein SDS Chuyén ciic tube eppendorf co chiia mdu vao d:i 30 phut Ly tain vong 20 phiit c› tfic d6 12.000g, chuyén dich ndi sang mot tube sach lanh, sau dfi chuyen vao d:i it nhdt phiit Ly tain vong 20 phiit fi tfic dfi 16.000g, nhe nhang loai bé dich n6i Rica can l¾g l6n big 500 pl cfin 700 a 10 Dé cac dng khfi tu nhien ngoai khfing hoac ly t6m ch6n khfing 11 xung 100 Ji1 TE vao mii tube de hoa tan ADN, béo quén ADN da t:ich chiet tit lanh sdu —20 dén -30' C cho din sit dung -Tién hénh phén ii:rig PCR.+Tién hanh ky thu8t PCR :Trinh tu moi xufii va moi nguoc duoc chpn dé nhdn ban gien dac hi(u cua Ascaris la: - NCS moi xufii; 5' GTAGGT GAACCTGCGGAAGGATCATT3 — NC2 moi nguoc; TTAGTTTCTTTTCCTCCGCT-3’ - dNTP : 2,5 pl - Buffer :2,5 p1 - Primer :2.0pl - Taq :2 dv(don vi) - ADN :5.0 pl - H O cho du 25pl +Chu trinh nJi ie’t (Dieu ki(n t6“i cua phén ring) -Bufic 1: 94'C phiit -Buoc 2: 94'C 30 gi6y -Bu‹ic 3: 60'C 30 gi6y x 30 chu ky -Bufic 4: 720C 30 gidy -Bucic 5: 720C phiit -Duy tri sén ph6m o 250C -Dién di Icie m tra sin phém PCI.- Sau thuc hi(n phén ring PCR tren in:iy lu6n nhiét xong, ta tién hanh die:n di kiém tra sén ph6m tr6n thach agaro néing d0 2,5 o moi truéng dién di TBE 0,§Ro co chiia ch6t nhuom m6u ethidium bromide v6i cu6ng d0 dong di(n 120 mA 30 phiit Sit dung marker chudn de kich thucic sén pham Doc ket qua dien di ducri dén tit ngoai thay giun diia ngu©i cho mot bang va giun dua lori cho mot bang co kich thuc›c bring va bang 980 bp San chup énh sin phdm di(n di tren Gel bang in:iy chup :inh ky thuttt s6 GDS-8000 -Sii dung enzym fI‹zeIII cii san pham PCR cua giun diia de’ tim su khéc biét giii:a hat loéi giun néi trén 5:in pham PCR ciia giun diia sau kiém tra da co ADN, ta tién hénh sit dung enzym HaeTD de cat sén phd m PCR v6i thénh ph ti n phan ring gfim: -H O : 24,5 ¿1 —Buffer : 5pl -Enzym If‹ieIII 0,5p1 -ADN : 20pl Dié u ki n tot in cho phén u:rig.- o’ nh’iét d0 370 C 16 gici, sau dfi di(n di, H kiém tra sén pMm tren thach agaro nGng dd 2,5Wo moi truéng di(n di TBE 0,59o co chiia chat nhufim mdu ethidium bromide v‹5i cuérng dfi dong di n 120 mA 30 phiit Sii dung marker chutin dé kich thuoc st ph9m Doc kit qua dieu di du6i dén tit ngoai th6y: - Giun dfia lpn cho bang chiem 79,319s ( v6i kich thuoc c:ic bang la: 610bp va 230bp va 140bp) 20,68 to giun dfia lori cho big ( v6i kich thucic cac bring la: 610 bp, 370bp, 230bp va 140bp) - Giun dfia nguéi cho big chiem 84,2Wo ( viii kich thucic c:ic bring la: 610 bp va 370bp) va l5,89o cho big ( v6i kich thuoc cac big la: 610 bp, 370bp, 230bp va 140bp) Sau dfi chup anh san pham di(n di trén Gel big in:iy chup th kj thu t so GDS-8000 -Xu: Ij st liéu vé hinh énh.- teo phuong phap thfing ké sinh hoc, ph6n mhm EPI INFO 6.0 va in:iy anh ky thu9t so GDS-8000 4.KET QUA 4.I.Tinh hinh nhiem giun tai diém nghién ciiu: 4.1.1 Tinh hinh nfiiém giun tai xii Phu:eng Trung Bang Ty lé nhiem giun tai xa Phuong Trung XN Ke" t qué xe“ t nghi(m duong Don nhiem tinh Giun moc Giun dfia Giun toc SL ( o) PL (to) SL 172 45 26,16 26 15, ('7n ) SL 4,66 (to) Dfia Toc SL (to) 11 6,39 Diia Moc Toc Moc SL (to) SL (to) 0 0 Ty Ie nhiém ino;t loai giun th6p, giun dfia chi chiém 15,119o, giun toc chiém 4,66 to \/a kh0ng g)p truéng hpp nhiem giun moc néo Ty 1( nhiem phfii hpp hai loai giun th6p va chi gap giun diia nhiém v6i giun toc chie‘in 6,39o (Big 1) 4.1.2 Z'inf i hinh nf iiém giun liii xzi f 'fiuiig Cliiiu B rig Ty 1( nhiem giun tai x5 Phung Ch6u N K6t qu:i xét nghi(m dgt 74 53 71,62 Da nhiém 17 22,97 12,16 0 21 28,37 0 Chu yéu la nhit:in giun diia vé giun toc, ty ie don nhiém véii giun diia chi6m 22,97Ro, giun toe don nhiém chi6m 12,16Ro W Vi5i di6u tra cr xa Phuong 8,1