S6 5-2012 T»P CHi P H 6 N 6 C H 6 N G BgNH S6T RET VA CAC BgNH KY SINH TRUNG 3 5
AP DUNG KY THU^T PHAN
ITNG C H U 6 IPOLYMERASE (PCR) DE PHAN BIET AU TRUNG GIUN M 6 C Vdl Au TRUNG GIUN MO TAI M Q T XA MIEN NUI TINH PHU THQ
Hoing Van Tan, Nguyin Thi Hinrng Binh, Nguyin Thi Hdng Nggc, Nguyin Hing Hgnh,
Nguyin Thi Hiin. Vi Thj Lim Binh vi CS.
Vien Sol rit - Ky sinh triing - Con triing Trung uong.
Tdm tdt
Sau khi nudi cay 33 mau phan cd trimg gidng vdi trimg giun mdc, giun md. Sau 7- 10 ngdy chung tdi thu hdi au ttiing cho vdo tube 1,5ml ii tien hdnh tdch chiSt ADN cua timg mau vd sir dung ky thudt PCR de gidm djnh 4u triing giun mdc va au trimg giun md. K^t qud dien di san pham PCR cho thay au trimg giun mdc cd kich thudc bang bdng 690bp, giun md cd kich thudc bdng bdng 870 bp. Trong nghien cxni nay, chiing tdi xac djnh dugc 28/33 trudng hgp nhilm giun md dcm thudn(chi6m 84,84%) va 5 trucmg hgp nhilm phdi hgp giun mdc vdi giun md (chiem 15,16%), khdng gdp b?nh nhdn ndo nhiem giun mdc dcm thudn.
1. D^T VAN DE
6 Viet Nam, benh giun mdc, giun md Id mgt benh giun khd phd bien, benh luu hdnh d nhilu tinh thanh gong cd nude, dac biet Id cdc viing trdng mau cd dimg phan tuoi khong u de bdn rau. Giun mdc va giun md cd dac diem sinh hgc, djch te, gdy benh va phdng chdng benh tucmg ddi gidng nhau nen dugc ggi dudi ten chung Id benh giun mdc. Ty le nhiem va phan bd cua hai loai giun ndi tren khdng ddng dlu, tuy theo dac diem thd nhudng, tap quan canh tdc, ve sinh mdi trudng[4].
Nguyin Phan Long, Kilu Timg Lam (1975) cho bill d miln Nam vd miln Trung Nam Bd vimg ddng bdng ty le nhilm giun mdc, giun md chiem 53%, vimg ven bien 68%, trung du Id 61%, cao nguyen 47%. Theo cdc tac gid, 6 nude ta cudng do nhilm giun mdc nhin chung Id nhe, da sd cdc vung dilu tra sd tning trong mdt gam phan trung binh <1000 tnlng[2].
Theo dilu tra cua Bd mdn Ky sinh triing, Trudng Dai hgc Y Ha Ngi, 1997 d miln Bdc, ty le nhiem giun mdc d da sd cdc dilm dilu tra la 30 - 60%. Vimg ven biln ty \i nhilm cao hon cd 67% sau dd trung du cd ty le nhilm Id 64%, cdn vung nui Id 61 %I4].
Hoang Thj Kim, Nguyin Vdn Dl vd ctv(1990) cho thdy d cac tinh Bdc bg, nam gidi cd ty le nhiem giun mdc 59% va nO gidi 74% cdn cdc tinh miln Nam, nam gidi chilm ty le 56%, ntr gidi 78%, trong sd dd cd 2/3 Id nhilm giun md[l].
Hinh thi du trimg giun mdc thay ddi theo timg giai d o ^ phdt triln ciia du trimg vd thuc te rdt khd phdn biet voi cdc du trung tucmg tu. Theo Kilu Tiing Ldm, khi nghiin ciiu nhihig
3 6 s6 5 -2012 TAP CHi P H 6 N G CHtfNG BgNH S6T RJT vA CAC BgNH k» SINH TgUNG
hinh dang ciia au triing thugc hp Ancylostoma ndi chung rlt khd phdn bift hinh the timg lodi.
Theo OS. D 5 Duong Thdi, vi$c phdn loji kn triing c4n phdi dga vdo cdc djc tinh miln djch vd dimg cdc huylt thanh khdng giun mdc dl xdc djnh lofii kn triingIS].
Theo Jozefzoon LM, Oostburg BF 1994, dl chin dodn b$nh giun mdc vd giun md ngudi ta dua chu ylu vdo -nit nghiem de tim tning trong phdnjlO]. Tuy nhiSn, vi^c phdn bi?t tning cua hai lodi giun ndy v5n cdn khd khdn do v9y ngudi ta thudng nudi cAy phdn theo ky thujt ciia Harade - Moti Ai phdn bi^t chiing thdng qua kn triing tu6i ba (L3) [7], hodc sii dyng ky thudt PCR de nhdn bill kn triing thdng qua ADN ddc thii cho tirng lodi[7] [10].
Tai-Soon Yong vd CI V. 2007 sir dgng ky thudt PCR dl xdc djnh giun md, giun mdc tt- udng thdnh. Sdn phim PCR sau khi di$n di cho thiy giun mdc cd kich thudc bdng Id 690 bp cdn giun md cd kich thudc bdng Idn hon vd bdng 870bp[13]. Mdc dau vdy, cho den nay d Vi?t Nam chua thiy tdc gia ndo sir dgng ky thu§t PCR dl phdn bi^t lu triing giun mdc vdi lu trung giun md. Vd vdy trong nghidn cim ndy, chyng tgi hy vgng b6 sung mOt phuong ph^
mdi trong djnh lo^i lu triing giun mdc, au triing giun md tgi Vi?l Nam.
2. T H 6 I G I A N , D I A DitM, D 6 I TirpNG VA PHlTONG PHAP NGHIEN CtTU 2,l.Thdi gian nghien ciru:
NghiSn ciiu dugc tiln hdnh trong giai doan 2011 - 2012.
2.2. Dja diem nghiSn ciiu
- Thu thdp mau: Xa Long Cdc, huy?n Tan Scm, tinh Phu Thg.
- Phdn tich mdu: Labo Sinh hgc Phdn tii Vi$n Sdt rdt - KST- CT.TlT 2.3. Doi tugng nghien ciru:
- Giun mbc Ancylostoma duodenale (Dubini, 1843); (Creplin, 1845) nhiem d ngudi.
- Giun md Necatoc americanus (Stile,1902); (Stiles, 1906) nhiem d ngudi.
2.4.Phirorng phdp nghien elhi
Dimg ky thudt PCR dl phdn bi$t lu trimg giun mdc vdi lu trimg giun md theo Tai - Soon Yong - HoLee vd ctv, 2007(13].
2.4,l.Thu th^p mau bfnh phdm
Xet nghiem phdn theo phuong phdp Kato - Katz (WHO) dl tim bgnh nhan giun mdc, giun md. Tien hdnh nudi cly lu trimg giun mdc, giun md theo ky thudt thudng quy cAa Vi$n Sdt rlt - Ky sinh trirng - Cdn trimg trung uong, nudi cho lu trimg phat triln r6i thu th|p lu triing Ai tdch chilt ADN.
a o a - m i z TAP CHI PHONG CHONG BgNH SOT RET VA CAC BENH KY SINH TRUNG 3 7
2.4.2. Bdo quan vd phan tich mau:
Au triing giun sau khi tliu thdp dugc dua vdo dng enppendorf 1,5ml. ghi ngay thu mau, ngudn gdc va bao qudn mau d til l ^ h sdu (-20''C den - 30 ''C) cho d^n khi sir dung.
Sir ly so lieu: Sir dung phan men EPI INfO 6.0 vd he thdng chup dnh ky thudt sd GDS - 8000.
2.4.3.Tdch chiet ADN:
ADN dugc tdch chiet bdng bd sinh phdm (KIT) theo hudng dan ciia nhd sdn xult. Tdm tdt cdc budc nhu sau:
1 Au triing mdi mau dugc thu thap vdo timg dng 1,5ml.
2. Them vao moi dng 180 ul Buffer ATL.
3. Them vao 20 |jl proteinase K sau do Irgn 15 gidy 5. U qua dem d nhiet dg 56"C
6. Them 200 ^1 Buffer AL, ddng ndp dng va trgn 15 gidy.
7. Them vdo moi dng 200 al cdn tuyet ddi lanh ddng ndp vd trgn 15 gidy, sau dd u d nhiet do phdng trong 5 phut (nhiet dg phdngl 5 - 25 "C).
8. L) tdm nhanh cho nude tre'. thdnh ddn het xudng day ong
9. Chuyln djch sang dng cd cgt Igc ddng ndp vd ly tdm SOOOv/phiit x 1 phut.
10. Md dng. them vao moi tube 500 dl Buffer AWl day ndp dng rdi ly tdm 8000v/ phut X 1 phut.
11. Thu col lgc chuyln sang dng sach khdc idi them vao 500 fil Buffer AW2. ddng nap dng rdi Ij tam 8000v/ phut x IphiU.
12. Chuyln cgt loc sang dng sach khac rdi ly tam 14000 v /phut x 3 phiit, dl cdt lgc dugc khd hoan todn.
13. Chuyln cdt lgc sang dng 1.5 ml sach vd them 50 pi nude cit, ly tam nhanh 14000 v/
phut X 1 phut, bd cpt lpc di thu ADN cdt til lanh sdu din khi sir dung.
2.4.4.Tien hanh phan ihig PCR:
- Ky thuat PCR dupe thuc hien theo quy trinh cua Tai - Soon YONG vd cs, 2007 [13], - Trinh tu mdi nhu sau:
NC5 mdi xudi GmAD); 5 TGCGAAGTTCGTTTCGCTGAGCT-3' - NC2 mdi ngugc (jmNA); 5" CGTTAACATTFTATACCTGACATAC-3'
Mdi ngugc GhTsp); 5 TTATGTGCCACAAATGAAGA-3'
3 8 S65-2012 TAP CHJ P H 6 N G C H 6 N G BgNH SOT Ref VA CAU B^wri Ki aiKn "^^.^
Thdnh phan phdn ling:
- dNTP 250 pM, 10% glycerol, 0,01gelatin vd 1 mM MgCL2. Primer 125pmol, Taq 1 don vj, ADN khudn 5 pi.
- H; O cho dii 50 pi hon hgp cdc chit phdn img.
Chu trinh nhif t (Dilu ki?n t6i uu cOa phdn irng) - Budc 1: 94°C trong 5 phdt - Budc 2: 94°C trong 1 phiit -Budc 3: 52"C trong 40 gidy
-Budc 4: 72°C trong 90 gidy x30chuky -Budc 5: 72°C trong 15 phiit.
-Duy tri sdn phim d25°C.
Di^n di ki£m tra san phim:
Sdn phim PCR dugc kilm Ua bdng ky thujl di?n di, nong dg thach agarose 2,5% trong mdi tnrdng TBE 0,5X vd chit nhugm mdu ethidium bromide vdi cudng dg ddng di?n 120 mA Irong 30 phiit. Su dung thang chudn lOObp dl do kich thudc sdn phim. Dgc kit qud dign di dudi din tir ngoji rdi chup dnh sdn phdm difn di bdng h? thing chgp dnh ky thujt sd GDS - 8000..
3. K £ T QUA VA BAN LU^N
a.l.Tinh hinh nhilm giun theo nhom tudi tai diem nghien cull Bing /: Ty If nhiem giun theo nhdm tuoi Nhdm tuoi
<5 5 - 9 10-15
>15 Tdng sd
So xet nghifm
1 8 24 198 231
Kit qua xet nghifm So dirffng
tinh SL
0 0 2 72 74
(%)
0 0 8,33 36.36 32,34
Nhiem mgt lo^i giun SL
0 0 2 63 65
(%)
0 0 8,33 81,81 28,14
Nhiem hai I09I giun SL
0 0 0 9 9
(%)
0 0 0 4,54 4,54
Nhilm ba loai giun SL
0 0 0 0 0
(%)
0 0 0 0 0 Nhdn xet:
- Ty le nhiem giun chung trong nhan din t^i diim nghien curu chilm 32,12%.
- Ty 1§ nhiem giun 6 nguoi tren 15 tu6i chilm 36,36%(bing 1).
S6 5-2012 T»P CHi P H 6 N 6 C H 6 N G BgNH S6T RET VA CAC BENH KY SINH TRUNG 3 9 Bing 2: Ty If don nhilm vd da nhiem cdc loai giun
Sd xet nghiem
231 Tyl?(%)
Ket qua xet nghi?m Sd ngudi nhilm mgt lodi giun
Dua
3 1,29
Tdc
16 6.92
Mdc 46 19,91
Sd ngudi nhiem nhieu lodi giun DHa Tdc
1 0,43
Diia Mdc
1 0,43
Tdc Mdc 7 3.03
Diia Tdc Mdc
0 0 Nhdn xit;
- Ty 1? nhilm mgt lo^ giun dua rdt thdp (chilm 1,29%). trong khi dd ty 1$ nhiem giun mdc chilm 19,91%(bdng 2).
- Ty le da nhilm giun tai dilm nghien cihi ciing khdng cao chi cd 9 benh nhan (chilm 4,54%)(bdng 2).
Bing 3: Ty If nhilm giun moc theo gidi tinh
So xet nghlfm
231
So dirong tinh
74
Tyif(%)
32,34
Gidi tinh Nam
Sl du-ong tinh
38
Ty If (%) 51,35
Nii So duong
tfnh 36
Tylf(%) 48,65 Nhdn xet:
- Ty le nhilm giun moc tai diem nghien cijru li (23,80%).
- Ty le nhiem giun moc giiia nam va nii (51,35%) so vai (48,65 %) khac nhau khong CO y nghla th6ng ke (voi p < 0,001)( bang 3).
3.2.Tinh hinh nhiem giun m6c vi giun mo
TiSn hinh nuoi cay 33 miu phin co tnhig giong voi tning giun moc va trung giun mo.
Sau nuoi ciy tir 7 - 10 ngiy thu thip iu trung &k tach chik ADN cho ky thuat PCR. Sin phim PCR dugc phin tich bing ky thu^t di?n di cho thiy co hai kiSu bang. Kieu bang chiem da so (28/33) mau co kich thuac la 870 bp vi dung bing kich thuac bang ciia giun mo Necator americanus (hinh 1). KiSu bang thii 2 c6 kich thuac nho hon va bang 690bp, tuang ling voi giun moc Ancylostoma duodenale (hinh 2). Ket qua nghien cuu niy tuong tu vai nghien ciiu cua Tai - Soon Yong va ctv, 2007[13]. Ket qui cho thay benh nhan tai diem nghiSn ciiu da so la nhiem giun mo 28/33 (chi^m 84,84%). Co 5/33 benh nhan bi nhilm phoi hgp ci hai loai
4 0 565-2012 TAP CHi P H 6 N G C H 6 N G BgNH SfiTRgTVA CAC BgNH KVSlNHTKiJNG
giun md vd giun mdc chilm 15.15%. Trong nghien ciru ndy chiing tdi khdng gdP bfnh nhin ,1' ndo nhiem giun mdc don thuln.
Id 8711 bl
lliilh I: Hinh .uili Jifi: ,li M//I /'!""" ' ' ' '> ' " " ' " ' """>•• '-•'"" ' " "
f . i f lOI 1. 1 "'. -4. .^.7. S. '). 1 I l:> C;K- bant! ma au liiing giun iiw vcri kich Ihudc 1 bp
i o l b ialliaiit ilo "inbp
I 2 3 4 5 6 7 8 ') 10 I I 12 1.1 U I 5 l b 17
8711b P 6Wb P
==^
Hinh 2: Hinh dnh diin di san phdm PCR ciia benh nhdn hj nhiim giun 1110 vd hinh nhdn nhiem phoi hgp hai logi giun md vd giun moc
Cgt 3, 8, 9,10 Id hinh dnh nhiem phdi hgp hai logi giun mdc \a giun md - Giun mdc cd kich thudc bdng bdng 690 bp.
Giun md cd kich thudc bang bdng 870 bp.
Cdt sd 17 Id thang do 100 bp
4. KET LUAN
Bdng ky thudt PCR ta hodn todn phan bift chinh xac dugc au triing giun mdc {Ancylostoma duodenale) vd au triing giun md {Necator americanus ).
S65-2012 TAP CHi PHONG CHONG BfNHS6T RET VA CAC BgNH KY SINH TRUNG 4 1 TAI LIEU THAM KHAO CHINH
1. Hodng Thi Kim.Nguyen Vdn Dl. Nguyen Thj Dang. Nguyin Thj Mai. Hodng Van Tdn, Nguyen Thi Diic (1991-1996^, "'Nghien ciiu hieu qud dilu tri giun mdc cua mgt so thudc lien quan voi cudng do nhiem va chung loai giun". Ky yeu cdng trinh nghien cuu khoa bgc ", Tap II. phdn ky sinh triing va con triing y hgc. Vien sot ret - Ky sinh triing- Con triing trung irong. tr: 38-47.
2. Nguyen Phan Long. Kilu Tiing Lam. (1975), "Mgt s6 dac diem ve tinh hinh nhilm giun md vd giun mdc d Bdc Vift Nam". Ky yeu cdng trinh nghien ciru khoa hgc 1975. Vien sot ret - Ky sinh Irirng - Cdn triing Trung ircmg. tr: 189-197.
3. Du an Phong chdng giun .idn Viil Nam WHO (2002). Tdi lieu Tdp huln chuyen mdn ve cdc benh giun truyen cho dat.
4. Nguyin Thi Minh Tam, Pham Hodng Thi. Pham Vdn Than. Pham Tri Tue, Hodng Tan Dan. Bd mdn Ky sinh triing. Trudng Dai hgc Y Ha Ngi, 1997.
5. Dd Duong Thdi, Trinh Vdn Thinh (1976). Cong trinh nghien ciru Ky sinh triing a Viet Nam. Tap I, Nha xudt bdn Khoa hgc vd Ky thudt, tr: 141 - 160.
6. B. Zahan, T.Li. B. Xiao, F.Zheng, and J.M. Hawdon (2001), "Species - Specific Identification of Human Hookworms by PCR of the Mitochondrial Cytochrom Oxidase
\%ent". Journal of Parasitology Allele: pp. 1227- 1229.
7. Monti J.R.; Chilton N.B.; Bao - Zhen Q.; Gasser R.B (1998). "Specific amplification of Necator amerccinus or Ancylostoma duoenale DNA by PCR using markers in ITS -1 rDNA. and its implications". Molecular and Cellular Probes. Vol 12, Number 2, April, pp 71-78(08)
8. Yukio Ychide (1996)."Morpholocation differences between Ancylostoma duodenale and Necator americanus in the fouth stage". The Journal of Parasitology: Vol. 52. No.l (Feb.. 1966). pp: 122-126.
9. Jhn M. Hawdon, Medical Heminthology Laboratory, Yale Univercity School of Medicine, 501 LEPH. 60 college St. New Haven, Connecticut 06520 (1996)
"Differentiation between the human hookwom Ancylostoma duodenale and Necator americanus using PCR - RFLP", The Journal of Parasitology. Vol 82. No 4.
10. Jozefzoon LM, Oostburg BF (1994). "Detection of hookwonn and hookworm-like in human fecocultures in Suriname". Am J Trop Med Hyg Oct; 51 (4): 501 -505 11. Johanna M.de Gruijter, Lisette van Lieshout, Robin B, Gaser , Jaco J. Vewei, Eric
4 2 S65-2012 T»P CHi P H 6 N S CHfiNG BgNH S6T RIT VA CAC BgNH KV SINH TRUNS
A.T.Brienen Juventus B.Ziem Lawrence and Anton M.Pooiderman (2005). "Polymerase chain reation - based differential diagnosis of Ancylostoma duodenanle and Necator amerycanus infections in humans in northern Ghana", Tropical Medicine and International health. Volume 10. No 6 . pp: .574 - 580..'
12. J.J.Ver; D.S.S.Pit, L.van Lieshout,S.M.Baeta, O.D.Dery,R.B.Gasser and A.M.Polderman (2001), "Determining the prevalence of Oesophagostormum bifruicum and necator americanus infection using specific PCR amplification of DNA fromfaecal ssamphes". Tropical Medicine and Internatioal health. Volume 6 pp;726-731 septemper 13. Tai- Soon YONG, Jong - Ho LEE, Seobo SIM, jongweon LEE, Duk -Young MIN
jong-Yil CHAL, Keeseon S.EOM, Woon-Mok SOHN, Soon - Hyung LEE vd Han - jong RIM (2007), "Differential diagnosis of trichostrongylus and hookworm eggs via using PCR ITS -1 sequence", Korean journal of parasitology. Vol 45. No,l; tr: 69 -74.
DIFERENTIAL DIAGNOSIS OF LARVAE A. DUODENALE AND N. AMERICANUS BY PCR IN A MOUNTAIN COMMUNE OF PHU
THO PROVINCE
Hoang Van Tan. Nguyen Thi Huong Binh, Nguyen Thi Hong Ngoc Nguyen Hong Hanh, Nguyen Thi Hien. Vu Thi Lam Binh vi CS.
Abstract
33 fecal samples contained hookwormlike eggs were culttued for 7 -ID days. After that we collected larvae and put in to 1,5ml tubes. Then DNA were extracted for using PCR method. Electraphoresis of specificaly amplified PCR products of A. duodenale and N.
americanus from the eggs of each 690 bp and 870 bp respectively. The results of this study were shown that 28/33 cases were infected by sing N. americanus (contaened 84,84%) 5 cases were mix infected by A.duodenale and N.americanus (contaened 15,6%) non of any cases was infected by single A.duodenale.
Th»c sy. HOANG V A N TAN Khoa Sinh hgc phan tir. 04.5534765 Vi^n Sdt rdt - KST- CT TU
Cdn b$ phan bi^n T.S.LS Dire Ddo
Trudng Khoa Sinh hgc Phdn Tu Vien Sdt ret - KST- CT.TLT.
