Áp Dụng Kỹ Thuật Phản Ứng Chuỗi Polymerase (Pcr-Rflp) Để Phân Biệt Giun Đũa Người Và Giun Đũa Lợn.docx

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 5 n6m 2006 ddn th:ing 12 nam 2007

H8 N0 - 2 7


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
1.
T8n

Chis nhi in dc: tai:

Th.s.Hoéng Viln

2.

Co quan chin tri dé tai: Vi(n Sit rét- Ky sinh triing- Cm
triing Trung uong

3.

Co quan qu:in IQ de tai: Vi(n Sit rét- Ky sinh triing- C6n
trilng Trung uong

4.

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 5 t rét —KSTCTTU Th.s. Le Diic Dao
Vi n 5 t rét-KST- CTTU
Th.s. Ha Viét Vién
Vi(n Sit rét-KST- CTTU

5. Théi gian thuc hien:Tit thiing 5 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 trong

ITS-2 : Vung phién ma trong
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 do 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 thanh dé tai nay 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 trong
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 trong c:inn on moi su giiip do qiii b:iu do !
Ha N i, ngay 20 th:ing 8 nam 2007


MUC LUC
Tran8

PHAN A.TOM TAT KET QUA NPI BAT CUA DE TAI........................1

2.1..................................................................................................................Nghien
cilu trong nuoc.................................................................................................4
2.2..................................................................................................................Nghi6n
ciiu nuoc ngoai.................................................................................................5

3..T1-HJ GIAN,

! DIEM, DU TUDNG VA PI-I UDNG PI-IAP NG I-H EN

C’UU............................................................................................................................................8
i.i.wai gian nghién emu.................................................................................8
3.2.Di|a diem nghie‘n erm..........................................................................................8

3.3. r›oi twong nghién erm.............................................................................................................8
3.4..........................................................................................................................................................Phuon
g ph:ip nghi6n ciiu..........................................................................................................................8
3.4. I..Twist ké nghién cim..........................................................................................................9

3.4.3.......................................................................................................................C:ic
ky thuAt sit dung trong nghie’n c6u.............................................................8
3.4.3.1....................................................................................................................Thu
th8p mAu va béo qu:in mtl u...........................................................................8
3.4.3.2....................................................................................................................PhAn
bi I giun dfia nguoi va giun dfia ton bang dltu hi(u hinh th:ii....................8
3.4.3.3. PhAn bi(t giun diia nguoi va giun dua lpn bang ky thullt PG R.............8
4. 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. 1 5:in ph4m PCR ciia giun diia nguéi vé giun dua ton khi chua cat enzym
Haelil......................................................................................................................................16

4 .2.2.2.Siin pliam PCR ciia giun dfia ngiicri va giun diia ton da cat enzym
HaeIH.....................................................................................................................................................17

5. DAN LUAN............................................................................................18
5. 1 .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

6.....................................................................KET LUAN VG OE NGH !

20
TAI LIEU THAM KH 0.......................................................................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, trong 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 do
cat s:in ph±m PCR bring enzym HaeW vt tri 358 dfii vdi giun d fia nguéri cho ra 2 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 ra 3 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 trong nghién erm nay chiing Hi ph:it hi(n dune
14,7'7o giun dfia ngiiiii co 4 bring, trong 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 con 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 nay ta co the xtic d]nh dupe kha n:ing nhiém
chéo giun dua ton tren nguéii va nguoc lai.
SA
2. AP DUNG VAO THUC
N sinh hoc phdn tit ve ky sinh triing duérng ruot trong
Ung dung thuc tién tai c:ie labo
vi(c
chlln do:in phttn bi t giun dfia nguéii va giun dua ton.
3. DANH GIA DC TAI VIII DE CUONG NGHIEN CIJIJ DA DUtJC DUVET
a. Dt: tai da thqc hi(n diing ti6n do ( 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 min 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 trong 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 do giun chui 6ng in t, tac ru t do giun v..v..
Nufic ta la nuoc nong nghi(p, nam trong vilng nhi(t d‹ii gio miia co dint

ki(n khi 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 pho 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 ra c:ic bi(n ph:ip phong chong &nh thich
hpp nham lam giém ty I( va cuéng dfi nhiém giun trong 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 do ddn cu song t8p trung, diéu ki(n ve
’i truéng sfing quanh ta b) fi nhiém triing giun r8t
tr6m trong (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 6 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 con cao o ca 3 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 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 thanh tit ngu i va tir v I chu ton la tuong tu nhau, chu k sing ciia hai


loai giun nay giong nhau, do 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.
2.

TONG QUAN TAI LI E) U

2.1.Nghién elm trong nu‹ic
Dinh loai dna vao c:ic ddu hi(u hinh th:ii duoc :ip dung tit xa xua va r6t pho
bién cr nhiéu nucic trén the gi6i. Phuong ph:ip nay da dong gop to 1‹in vao vi(c
xac dmh thénh phtih loai trong 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 ngay tai thuc dia. Tuy nhien, phuong
phiip nay cling b6c 16 nhfmg han ché nh:It dinh trong nhfmg truéiiig hpp m6u
vilt khfing dii i hinh, m6u v6t khfing con nguyen ven, cac mdu vat co miic dfi
tuong dong cao v.v. . .thi viéc x:ic dinh thanh phan loai cling gap phai nhfmg
kho kh nh6t d]nh.

Do Duong Th:ii,(1971) khi nghién chin vé hinh the triing, au tiring va giun
truéng thanh thdy rang hi(n v8n con khfi kh , chua ph6n bi(t duoc gifia giun diia
ngufii va giun dfia Ion, do 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 trong phln loai, vita King dung
trong 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 thanh co sq
thay doi tuy 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 trong moi truéng béo t8n gfim nucic sinh ly vé glucoza, m6u sac
giun cling kh0ng ’ i cho c0ng t:ic dinh loai[4] .


Mot so tac gia lai dna véo v| tri lo sinh duc vfi lo bai tiet ciia giun c:ii de
x:ic dinh, nhimg c:ie dac diem nay thuéiig khong on dinh va co thay doi giiia
giun dfia ngucii A. /nmbrico/dc x va giun dfia lori A.xoom. Do vgy Krotor, Pham
Hoang The (1973) de xu6t khfing dna vao dac diém nay de dinh loai [1].
rnfit so” i dduuaa nguu6ii phhtéinn duuffiiii co‘ m6u thit hiinnhh
in cc::iinhh, nhimg man thit
nn

n
nay chi co trén mGt so giun (20Wo) Va chua x:ic dinh dupc tinh chdt lien quan
dén d[nh loai (DC Duong Thiii, Phijm Hoang Thé, 1973). So v6i tai 1i(u co
dien, nhfmg d6n lieu mcii va cdu tao cc quan sinh duc cua giun dfia khfing co gi

khéc
truoc va
’ i sai khéc v6i Hi lieu nu6c ngoai. Ngay ca nhiirig hinh thi: cua
khfing
‘u vlt eat manh ciing khfing sai khiic v6i ket qua nghién ciiu ciia thé gi6i (D6
Duong Th:ii va CS, 1973) do v8y ciing kh0ng thé dna vao nhfmg dac diém dfi dé
dinh loai [1J.
Nhfmg nghién ciiu ph6n loai big hinh th8 gifra giun dfia nguiii va
dfia Ion tru6c d6y chua thuc su co tinh thuyét ph9c cao, mat kh:ic nham
ra
trién vong nghién ciiu ve gen hoc trong tuong lai, Doén HQ Nh6n (1971) da bat
dan nghién ciiu the nhiem sac trén giun diia ciia nguéi. Tac gia da diing tinh hoan
cua
giun
duc
dé
t:ich
va
dem
‘
tthhéé
nhiém
dung d]ch camoy rfii nhuom Giém sa va
quan s:it. Theo t:ic gi:i, nhdn te" bao ciia giun dua co 24 nhiém sac the, mat
kh:ic hinh dang the nhiém sac giun dfia nguéi co hinh the timg dfii dang V, dang
chfim, dang que, dang chiiy that [1J.

2.2 Nghién emu ngoiti nirdc:
Da nhiéu nam qua, nhfmg tranh ludn ve giun diia tir nguéi Ascaris
lumpricoides (Linnaeus, 1755) v6i A.xoom (Goeze, 1782) tit lori co phai la ciing

loai hay la hai lo:u ri6ng biet da dune hang loat c:ic nghien ciiu kh:ic tim den dia
chi ciia c6u hfii nay [9,14. . .]. V6i nhiing dieu tra cat ngang, chfing minh giun
dfia ct ngiiéi co thé nhiém sang Ion va nguoc lai, nguéi cling co thé nhiém giun
dfia tir
gia kh:ic dE nghien cilu
vilt chu Ion [14,16,27]. % tain ro vdn de nay, met ‘
t
téé
cc
4


hai loai giun nfii tren dna vao hinh the: [16, 17, 25, 26J, hinh the ciia du triing giun
diia r6t don gién va nhfi, do v y viec xéc d)tnh chinh x:ic loai la gap nhiéu khfi

khan [24,26].
Mfit so nghién ciiu dira vao rang ciia giun dfia (Linnaeus,1758;
Sprent ,1952; Bv W.Crewe va David H.Sminth,1970). Theo tiic gié Linnaeus
1758, rang ciia giun dfia nhfi, riing co hinh tain gi:ic nam ci phia trong va cufii bé
m6i cua giun [17].

4


Sri S.Abdulrachimax va Lie kian Joe (1954) [25]. ciing dGng

’ véii Sprent,
rang cua hat 1oiii nay la khfi x:ic dinh, tu nhieu gfic dfi kh:ic nhau cua rig, cé ve
hmh th:ii cling nhu ve kich co nhieu khi khfing
t:1c gia, rang ciia

“’

giun dua nguéi co chiéu diii 2-7p (thuimg thuéng 4-5p) va chi6u rfing 2-6 p
(thuéng thu rig 4-Sp). Trong khi dfi giun dfia tit lpn rig co chiéu dai 2-11 p
(thuéng thuéng 5 p), chiéu rong 3—9 p (thuéng thuéng 5-6p). C:ic téc gié cho
rang rig ciia giun dna Ion thuimg l6n hon va co tht: nhin ro hon rang ciia giun
dtia ngu i khi soi du‹5i kinh hilt vi [25J.
Khi nghién ciiii 93 mlu giun dfia trong dfi co 50 mau giun dfia tit nguéi va
43 m6u giun diia tu Ion, t:ic gié chi th6y 38J50 mau giun diia tit ngui:ii co rig
rig ciia loai
trong dfi co 12 mdu co kiéu rang dit:n hinh con
26
kh:ic.
5 giun dfia Ion phiit hi(n 31J 43
nhfi kiln giun dua nguiii con lai 28 mtiu co kiéu rang ciia loai khac. Tuy nhién,
cling kh:i nhiéu truéing hpp rig cua giun diia
thu rig khfi nhin th6y dupe
do
co cfic hat nhfi bao phii.
diia Ion, r6ng gdn nhu nhin
con th6y ro hon [25].
Theo d:inh gi:i ciia Sri S..AbduLachimax va Lie kian Joe (1954), nhin
chung rig ciia giun dua ngir i co kich
rig giun diia ton va su kh:ic
bi(t vé rang cua hai loai giun noi trt:n la khfing ro ritng[25J.
Ngoai vi(c dna vao rang d6 ph6n bi(t giun d fia nguéi va giun dfia lori,
M. Ansel va M.Thibaut (1972) con dna vao mi ng giun dfia d6 dinh loai. Theo c:ic
tac gié, ID mi(ng cua giun dfia ton hinh luc gi:ie con giun dfia ngu i IO mi(ng co
hinh tit giac [8].

Mot nhfim nghien ciiu kh:ic lai gi:inn dtnh Ascaris lumbricoides va
Ascaris sunni bring c:ic protein. Trong qu:i trinh nghit:n ciiu, céc t:ic gié da ph:it
hi(n ra 6 protein dac trung cho giun dfia Ascaris lumbricoides ma kh0ng co o
giun dfia lpn. Theo c:ic t:ic gia thi 6 protein dac trung dfi 1é:1, 2, 3, 4, 5, 6
tuong ring 118kDa, 116kDa, 110kDa,105kDa, 25kDa vé l7kDa [29].
Bang ky thu t PCR, c:ie tiic gia sit dung enzym ph8n c6t gi6i han H aeOI,
cat sén ph PCR cua giun dfia. Sau dfi, c:ic tac gié sir dung ky thu I di(n di,
thdy giun diia lori co 3 big, trong khi do giun dfia nguéi cho ra 2 bang tir do tae
giii co thé nh(n biét duoc giun dua nguéi va giun dfia ton, thing qua sfi luong
bllng va kich thucrc ciia cac big khi soi du6i dén cqc tim. [27, 28].
Anderson 1994, big ky thu6t PCR cho thdy
giun dua lori nhiém tren nguéi. Did:u dfi céng lo ngai khi co nhiéu triing giun dfia
dwoc tim thdy trong ddt va co kha uang nhiém nhi6u hon néu nhu ph8n Ion dune
5


sit dung bon c6y o vuén va c6y n8ng nghi(p. Mac dii v y, su nhiém chéo trong
tu nhién la co gi6i han (Anderson va CS, 1997), khi téc gia khfing ph:it hi(n
ra tru rig hop nao ngu6i bi nhiém giun dua Ion ci lang Guatemala[7].
Nam 2004, nhfim nghién ciiu hpp t:ic gifia Dan Mach va Bangladesh
gfim Perter Nejsum, E. Davis Parker, Jane Frydenbeng. . . da ti6n hiinh ph6n
tich 135 m6u giun diia ci 5 qu6c gia kh:ie nhau trén th6 gi6i, gfim 71 m6u thu th p
tir nguéi va 64 m6u thu th6p tu ton, trong dc› ct 32 m6u tir 29 ngu i Dan Mach.
Khi ph6n tich 32 m6u nay bang PCR-RFLP thi co 27 tarring hop do bi nhiém
giun dfia co nguon gfic tit Ion n0i dia. Trong s6 nay co trén 809o dtfclc biet co
tiép suc v6i Ion hoac ph6n ton. Tit dfi ciic nha nghién ciiu ra khuyén ciio Dan
Mach ct quan tdm nhiéu den ciic bénh tu thfi [23J.
© Trung Qu c, nghién ciiu vé su thay doi gen ciia qudn the giun dfia
nguéi va lctn o ciing diém ph6n b6 WeIDong Peng; TJ.C Anderson; Xingquan
Zhu va Wkennedy 1998, da chi ra mot vtlh dC quan trpng la co r6t it gen bat

ngu6n giua qu6n thé KST nguéi va qudn thé KST lpn, mac d6o lJch sit tiép site
gifra nguéi va lori da co tit r6t 16u ci Trung Qu6c, dong théi qua nghien ciiu nay
c:ie téc gia ciing khfing gap triréng hpp nhic:in chéo nao tai diem nghién emu
[27].

6


3. THAI GIAN,

cfJlJ

!A DIEM, DOI TLfCJ NG VA PH LfO NG PHAP NGHIEN

(5/2006-12/2007)
3.I. Thdi gian nghién ciiu : 18
tthh:iiinng
3.2. DJa diem nghién erm:
Dia diem thu mdu: xa Phuong Trung, huy(n Thanh Oai va xa Phung Chdu,
huy(n Chuong My', tinh Ha Tdy.
— DJa diém phW tich m6u: Labo Sinh hoc Phln tit Vi(n Sét re’t- KST- CTTU
Khai quat mot so net v4 diém nghit:n elm:

a/ Xa Phirong Trung:
Xa Phuong Trung, huy(n Thanh Oai, tinh Ha Tay la mot xa nfing nghi(p,

co d8n sG 14 nghin d6n (theo thfing ké 3J2006), di(n tich lira nucrc la chu y6u
chiém 4J5 di(n tich canh tac, di(n tich trfi ng mdu chi chiém US dién tich canh
t:ie. Ngoiii v6n dt: lam nfing nghi(p, nhdn ddn o xa con co nghé phu lam non,
litm la va bufin b:in, kinh doanh nhfi.

b/ Xa Ph png Chfiu:
Xa Phung Chdu, huy(n Chirong My, tinh Ha Thy co di(n tich trfing m6u
chiém khoang 2J3 di(n tich canh I:ic.Trong nfing nghi(p, httu hit c:ic gia dinh sit
dung hai ngufin ph:In chinh la phs chufing va ph8n ho:i hoc. Ngufin ph6n
chufing ducic sir d9ng dé b6n lot la chinh va d6u quan trong ld ph8n chufing h ttu
hét dune ’ k‘ truc›c khi sir dung. Tuy nhieu thinh tho rig v3n con mOt vai gia dinh
con diing ph8n tuoi hoa nucic la dé tu‹ii cho ng0, bdu, bi va lira.

3.3. Dtii tiiong nghién emu:

— Giun diia nhiém ci ngu i Ascaris lumbricoides (Linnaeus, 1758)
- Giun dfia tit v t chfi lori Asc‹iris seem (Goeze,1782)

3.4. Phuong ph:ip nghién emu :
3.4.1. Thiét ké“nghién en:u •. Nghit:n c6u mo té cat ngang

3.4.2. CI

diroc chpn theo co m6u sinh hoc, tOi thiéu 30 mau mii
loai. Tong s6 60 mau, trong dfi co 30 mdu giun dfia nhiém o ngiréii vé 30 mdu
giun dfia nhiém ci v t chit ton.

7


3.4.3. Céc kj thuiit en: dung trong nghién cii:u.
3.4.3.1.Thu znclu riit rii hero quzin znzlu
-Thu thép min giun diia rigs:éi [Ascaris lumbrieo’ides).- (Theo phuong ph:ip
thuong guy ciia Vién S6t rét -KST-CTTU).

Xét nghi(m ph6n tim ngucii nhiém giun dua big phuong phép Kato
(Theo WHO, 1991), sau do tién hénh tay giun va thu th8p mdu giun dfia tu
nguéi bang dai ph6n tim giun dfia sau ufing thu6c t6y giun trong théi gian tit 1-5
ngay, neu khfing thu dune giun thi thfii khfing phai dai phln nhfmg ngay tiép theo.
giun dfiii:
giun dfia trén thi truéing hi(n nay co rdt nhic:u loai, tuy nhién
chiing Hi chon nhfmg thu6c dac hi(u v6i giun dua nhu thu6c pyrantel pamoate
l25mgJvién do Canada san xuat tien hiinh diéu tri theo hu‹ing din ciia nha san
xudt va duoc Bb Y Te cho phép lim hiinh tai Vi(t Nam. Ngo¾ ra chiing t0i con
sir dung thuoc da dupc Vi(n S6t rét KST- CTTU va Du :in Phfing ch6ng giun
san do WHO tar trcl diéu tri héng loat nhi6u nam tai dia phuong dfi la thucic :
Mebendazonle 500mg lieu duy nh6t
(Ph:ic do diéu tri. theo Du én Phfing chong giun s:in do WHO tai tro giai doan
2002-2005).

-Thu thép min giun diia trén vét ché low (Ascaris sunni).Tai dia diem mo lpn ciia dia phuong ( Phuong thiic thu m6u giun diia lori vé
bao quén m6u gi6ng nhu giun diia nguéii)
-ftzio quiin mzlu: (Béo gum mdu theo theo Anderson va CS, 1993a)
Giun sau khi thu th p dupe riia big nu6c mufti sinh ly 0,9J9o, sau do cho
vao lo co dung 'cdn70’C, nghi ro ngay thu
g6c Ion tir d6u va bao
mau,
quan mlu ’ tit llaannhh s6u (-20'C den - 30 0C)
dung.
cho dén

3.4.3.2. Phén biét giun diia nguéi vé giun diia low 6 rig dia hiéu hinh théi
Phuong ph:ip d]nh loai big hinh thé co sit dung kinh hip, kinh hién vi dfii

pha de quan sat va chup énh nghi hinh.

Diing thv‹ c do co chia vach, do chi tiét timg giun, lam tiéu bén phén mieng
de quan sat chi tiét rig va so sanh gifra hai loai Asccaris lumbricoides, Ascaris
srom. (Tiéu bén lam theo ky thu t thuéng gut ciia Vi(n S6t rét-KST-CTTU).

3.4.3.3. Phén biét giun diia ngu:or vd giun dii a foci bfing I:j' tfiunt f'CR
Ap dung ky thu(tt phén ring chuoi polymerase ph6n tich tinh da hinh dfi dai doan
philn cat gi6i han (PCR- RFLP) theo Xingqua Zhu va CS,1998.


-Dung en, trang thiét 6[ vé hot chlt.+Dung en vé trang thiét 6[.•
-Mfit bfi micropipet loai Gilson, in:iy khu:1y tu, in:iy lac (Robolab). .
—Tube eppendorf: 1,5 ml, 0,5ml. . .,d6u tip loai: 10 I, 200 .l, 1000 .1.
—Tu shy, tit lanh, ly t6m i6c dfi cao.

‘

• Bufing PCR
- M:iy lufin nhi(t
• Thiét bi bufing phiin tich st ph6m
Dén tir ngoai soi gel, lo vi song; M:iy dién di + bG ngufin. .
- M6t micropipet loai 5-50 . l; d6u tube loai: 10.1, 200.1, 1000 .1
- Hé thong in:iy chup énh va ph8n tich kj thu6t s6 GDS-8000.
+Ho:i ch6t:
• Ho:i chat diing dé t:ich, tinh khiét ADN:
- Dung dich t:ich EB (extraction buffer)
- Cfin ethanol 700 99', lanh.
- TE(Tris-base + EDTA), Kac 8M
• Ho:i chht diing cho ph:in ting PCR
- dNTP,(dATP, dTTP,d CTP, dGTP)
- Taq polymerase, mii

- Dem PCR10X
• Ho:i chit diing cho dien di
— Tris-base, axit boric
- EDTA, agarose gel
— Loading buffer
• Enzym ph6n cat HaeD
- Enzym HaeW
- Dung dich d(m ph cat.
• Dung dich nhufim ADN
- Ethidium bromide 10mg/I.

-T‹icli chiét SDN.- (AND duoc t:ich chiet tit gien dich ITS-1 va ITS -2)
T:ich chiét ADN ciia ‘u v6t theo phuong ph:ip ciia Collin va CS (1987),
co diéu chinh cho phu hpp v6i timg d6i tupng nghién ciiu torn tat c:ic buoc nhu
sau:
1. U dung dich tiich chiet (EB) ci 650 C trong 30 phiit de bdt hoat enzym.


2. 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 khi m6u v t o d:Eng hon dich té bao.
3. U c:ic tube eppendorf co chiia md u cr 650 C trong 30 phfit.
4.Them vao moi tube 30 pl Kac 8M de kit tiia philc hpp protein SDS
5. Chuyén ciic tube eppendorf co chiia mdu vao d:i 30 phut.
6. Ly tain trong vong 20 phiit c› tfic d6 12.000g, chuyén dich ndi sang mot tube
sach.
lanh, sau dfi chuyen vao d:i it nhdt 5
phiit.
8. Ly tain trong vong 20 phiit fi tfic dfi 16.000g, nhe nhang loai bé dich n6i.

9. Rica can l¾g l6n 1 big 500 pl cfin 700 a
10. Dé cac dng khfi tu nhien ngoai khfing khi hoac ly t6m ch6n khfing.
11.
xung 100 Ji1 TE vao mii tube de hoa tan ADN, béo quén ADN da t:ich
chiet trong tit lanh sdu —20 dén -30' C cho din khi 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; 5 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 trong 5 phiit
-Buoc 2: 94'C trong 30 gi6y
-Bu‹ic 3: 60'C trong 30 gi6y x 30 chu ky
-Bufic 4: 720C trong 30 gidy
-Bucic 5: 720C trong 5 phiit.
-Duy tri sén ph6m o 250C.
-Dién di Icie m tra sin phém PCI.-


Sau khi 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 trong moi truéng dién
di TBE 0,§Ro co chiia ch6t nhuom m6u ethidium bromide v6i cu6ng d0 dong di(n

120 mA trong 30 phiit. Sit dung marker chudn de do 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 nhau va bang 980 bp. San do 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 ra su khéc biét
giii:a hat loéi giun néi trén.
5:in pham PCR ciia giun diia sau khi kiém tra da co ADN, ta tién hénh sit
dung enzym HaeTD de cat sén phd m PCR do 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 trong moi truéng di(n di TBE 0,59o
co chiia chat nhufim mdu ethidium bromide v‹5i cuérng dfi dong di n 120 mA
trong 30 phiit. Sii dung marker chutin dé do kich thuoc st ph9m. Doc kit qua
dieu di du6i dén tit ngoai th6y:
- Giun dfia lpn cho 3 bang chiem 79,319s ( v6i kich thuoc c:ic bang la:
610bp va 230bp va 140bp) con 20,68 to giun dfia lori cho ra 4 big ( v6i kich
thucic cac bring la: 610 bp, 370bp, 230bp va 140bp).
- Giun dfia nguéi cho 2 big chiem 84,2Wo ( viii kich thucic c:ic bring
la: 610 bp va 370bp) va l5,89o cho ra 4 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 1. 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, 1 8

('7n ) SL

4,66

0

(to)

0

Dfia
Toc

SL

(to)

11 6,39

Diia
Moc

Toc
Moc

SL

(to)

SL

(to)

0

0

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 2. 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

9

12,16

0

0

21 28,37

0

0

6

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