Ket qua chung toi thu du'c;1C cling chi ra rang nong dQ ADN plioi thai tl/ do trong mau ng 0 9i vi clla m~ tang Can theo tuoi thai va phI,!. Tir bang k~t qua tren chung toi nh~n th~y non
Trang 1KHAo SATvA DINH LUQ'NG ADN PHOI THAI TV DO
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TV' 6 TVAN DEN 22 TVAN BANG PHV'aNG PHAP REALTIME PCR
Tri~u Ti~n Sang*, Tr~n Van Khoa*, Dinh Doan Long**
,
TOM TAT
(I) Ma dau: Vi~e phat hi~n ra su' ton t(,licua
ADN phoi thai tl/ do va te bao phoi thai tl/ do
trong huyet tu'dng ella me da rna ra mQt hu'ong
chan doan tnnrc sinh moi bang bi~n phap khong
can thi~p De djnh 1u'c;1.19 du'c;1C ADN phoi thai tl/
do trong huyet tu'dng cue me chung toi su dl,mg
phu'dng phap Realtime -PCR dl/a tren ky thu~t
TaqManPCRRealtime co SU dl:mg cac dau do
huynh quang Ky thu~t nay cho phep quan sat
du'c;1c cec tin hi~u huynh quang cue cac gen dich
va biet du'c;1c so ban copy cua chunga tung chu
kY nhan gen (II) Doi tu'c;1ngnghien oru: chung
toi su d1,lngla 60 the: phu dang mang thai tti
tuan th(J'6 den tuan th(J'22 clla thai kyo(III).Ket
qua: Nong dQ ADN tl/ do 0 tuan th(J'6 va tuan
th(J'7 co mQt so tru'ong hc;1Pkhong phat hi~n ra
sl/ co m~t clla gen DYS14chi phat hi~n du'c;1C gen
nay trong tat ca cac rl'~u du'dng tinh vao tuan
th(J'8 Ket qua chung toi thu du'c;1C cling chi ra
rang nong dQ ADN plioi thai tl/ do trong mau
ng 0 9i vi clla m~ tang Can theo tuoi thai va phI,!
thuQcvao tting ca the.
SUMMARY
INVESTIGATION AND CELL FREE
DNA QUANTIFICATION IN
MATERNAL PERIPHERAL PLASA AT
THE GESTATION FROM 6 WEEKS TO
22 WEEKS USING REALTIME peR
*HQc vi~n Quan Y
** Khoa Y Dugc - Dl;liHQc Qu6c Gia Ha N<)i
Chju tnich nhi~m chinh: TrAn Van Khoa
Email: tvkhoabio@gmail.com Dt: 01684552737
Ngay nh~n bili:5/10/2CI4
Ngily duy~t bili: 16/10/2014
(I) Introduction :Thc discovery of the existence of fetal cells in maternal peripheral blood has opened up a new direction for the early diagnosis of congenital anomalies by means
of non-interference To quantify fetal DNA circulating in maternal blood we used realtime PCR method based on TaqMan PCR technique using fluorescent probes This technique allows
to observe the fluorescence signal of the target gene ADN for us to know the number of copiesof each target gene at each time point of the PCR process (II) Objects, chemicals ADN research methods: We shall select the 60 pregnant women
in the 6th week to the 22th week of pregnancy Along with two negative control sample of 2 ADN pregnant women not yet having sex once 5 ml of blood is taken ADN conduct split ADN separated plasma free plasma DNA as the other sample (III) Results: Free DNA concentration at week 6 and week 7, there are some cases not detected the presence of the DYS14 gene was only detected genes in all positive samples in week 8 our results obtained indicated that the concentration of free fetal DNA in peripheral mother blood and increaseswith gestational age, depending on the individual.
Keywords: fetal DNA; reatime-PCR;
non-interference
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I D~T VAN DE
Vi~e phat hi~n ra ADN phoi thai til do trong mall ngo<;livi eua my la mQt buac phat tri~n mai eua khoa hQc, rna ra mQt hu6ng mai trong ehfm dmin di truyb truae sinh b[tng bi~n phap khong can thi~p N6ng dQ ADN xudt hi~n trong mall ngo~i vi cua my a
cae thai di~m khae nhau la khong theo mQt
Trang 2Y HQC VI$T NAM THANG 11 -56£)~C BI$T/2014
;u<;mgnong Q (1 cac twin thai 13 en 22
tan trong SOul H20 deiof'-
DYS14 TGGCGATTAAGTCAAAT GAPDH F
CCCCACACACATGCACT
DYS14 CCCCCTAGTACCCTGAC GAPDH R
CCTAGTCCCAGGGCTTT
Probe (FAM)_ TCTGCCTCCCTG
(HEX)_AAAGAGCT AGG
DYS14 "CTGCT(T AMRA)
Probe GAPDH AAGGACAGGCAACTTG
GC (BHQ1)
~ : lu~t, do vay d~ t3i iru va Iva chon diroc
::~'ridi~m lfiymau ngoai vi cua me d~ tach
! _!J);\ ph6i thai tvdo ia co mot Ynghia rfit 1611
:_-,)ngchan doan va sang loc truce sinh, tranh
"':'-=lJYco chAn doan nham am tinh gia khi
-~ i.b0ng tach duoc ADi.-.Iphoi thai
:~ f)~ dinh hrong duoc ADN ph6i thai hru
bh'1h trong mau me chung t6i dtIng phuang
;hip realtime PCR j\!,a tren ky thu~t PCR
T:!qMan su dVng dAu db hu5rnh quang Ky
±~t nay cho phep quan sat cac tin hi~u
t;l5-nh quang cua cac gen dich va eho ta bi~t
:C'.!yC s5 bfm copy cua timg gen dich (1ttmg
;!Kri di~m cua qua trinh PCR Do v~y chung
:.31th\!'c hi~n nghien CUll nay vai m\1c tieu:
?hat hi~n va dlnh tuqng ADN phoi thai W do
?0ng huy~t tuang cua m~ b~ng phuang phap
::~ealtime-PCR, xac d!nh thai di~m ADN phoi
:hai trong mau m~ du tan d~ tach duqc ADN
IJ. £)01 TU'Q'NG, HOA CHAT VA PHU'O'NG
PHAp NGHIEN C(YU
2.1 DAitU'qng nghien cu'U
a nghien CUll Clla chung toi, chung toi
~),rachQn thai di~m cac tubi thai tiI tuftn thu 6
c~n tuftn thu 22 cua thai ky eua 60 mau
nghien cuu am6i tuftn thai tiI tu~n thu 6 d~n
ru~n thu 12 la 60 mau duqc lAy Sml mrm
trong tAt ca cac tu~n nay, cac mau duqe xac
dinh chinh xac duang tinh vai gen DYS 14 va
duang dnh vai gen DYS14 (1 tuftn thu 12
Juqc h,ra chQn ti~p thea d~ lay mau djnh
.
tuan, Cac m~u nghien cirunay vua duoc chay
nhan gen tren gen DYS 14 vira duoc dinh
luong s6 ban copy tren gen DYS14 b~ng c~p m6i tren nhiern s~c th~ Y (gen DYS 14) va gen GAPDH lam nQichirng. TAt ca cac phu nfT nay diroc ti~n hanh lfiy 5ml mau toan ph~n va ly tam 5000 vong/phut trong 5 phut r6i lAy huy~t tuong Cung voi 2 mau d6i chung am lit 2 ncr giai chua mang thai va chua quan h~ tinh d\lc l~n nao duQ'c lAy 5 ml mau va ti~n hanh tach huy~t tuang va tach ADN w do trong huy~t ttrang nhu cac mau nghien Clru khac Olc mau nghien Clru duQ'c lAy t~i Trung tam nghien clm Sinh Y duQ'c hQc quan S\T - HQc vi~n Quan y
2.2 Hoa chAt nghien cU'U
H6a ch§t tach chi~t ADN: - Ethanol 70%, Ethanol 100%, Proteinase K, Nuac ck B<) kit Quiagen H6a chAt cho peR: PCR Reaction Mix DNA Polymerase, Reverse primer Forward primer, ?robe, NtTac cAt khu ion va vo trtmg Thi~t bi may moe: May I~c 6n nhi~t (Eppendorf-f)lfc) May Ii tam cao t6c (Hettich-f)lre), Buf,ng thao tac peR (USA), May Realtime PCR (RotoGen -Quiagene), May PCR ABl 9700 (Applied Biosystem), TlI h6t hor chAt (Han Qu6c), May do n6ng d<) ADN (Nano Drop-USA)
2.3 PhU'o'ngphap nghien cU'u:
Huy~t tuang sau khi ly tam ti~n hanh lAy 200ml huy~t ttTong tach chi~t b~ng b<)kit cua Quiagen ADN sau khi tach chi~t duQ'c hoa
139
Trang 3CHUYEN DE: HiNH THAI HQC
CCCCCTAGTACCCTGACAATGTATTCATTCTCAAGCAAAACATGGTAATTCAGTA
ACGTTGACTACTTGCCCTGCTGATCTGCCTCCCTGACTGCTCTACTGCTGTCCTGA AAAATGCGAATTTGACTTAATCGCCA
Hinh 2 Hinh anh trinh tu doan gen GAPDH
CCCCACACACATGCACTTACCTGTGCTCCCACTCCTGATTTCTGGAAAAGAGCTA
GGAAGGACAGGCAACTTGGCAAATCAAAGCCCTGGGACTAGG
Hlnh trinh W doan gen DYS 14 minh hoa
vi tri g~n m6i cua gen DYS 14 bleu th] bang
mau xanh, con vi tri duoc co mall do la vi tri
g~n cua ddu do probe Tuong W nhtr v~y tren
gen GAPDH vi tri duoc boi mau xanh Iii vi
tri giLnm6i cuagen GAPDH va vi tri boi mau
do la vi tri g~n ddu do probe
Qua trinh chay Pt:R duoc thuc hien theo
chu trinh nhir sau: Phan irng PCR voi tong
th~ tich phan ung lit 25ul trong do peR
master mix l Oul: Primer DYS14 (F, R,
Probe): (0,25:0,25:0,5)ul va primer GAPDH (F, R, Probe) : (0,25: 0,25: 0,5)ul; mroc: 8ul Chu trinh nhi~t 1 elm ky (95°C trong 10 phut); 55 chu ky (95°C - 1 phut, 60°C - 1 phut), eho may doc huynh quang i:t nhiet dQ
60°(' trong 1 phut, XLI' Iy s6 lieu bang phan mem chuyen dung ella may chay relatime-PCR RotoGen -Quiagen cia tich hop sfin va cac thuat toan
th6ng ke tren ph fin mem phan tich 56 li~u Excel Microsoft Ot1ice
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III KET QUA VA BAN LUA.N
3.1 K~t qua sau khi eh~y dU'()'Ogehu§n eua gen DYS14 va gcn GAPDH o'm~u ehuan pba loang o'cae nang dQ 104; 103; 102•
l_~~~~-;."qi~~?l.-;±;!S6- _·8:l2!!2!!iZ£!!I :,' '-_
gr-Hinh 3: Hlnb :lub plliill dcb duO'ug cbuiu cua geu GAPDH Hiub 4: lfulb ;lllh phau tich Quang ChU3l1 cua geu SRY
TiT k~t qua hinh anh tren nh?n thfty r~ng tin hi~u Clla gen GAPDH len d<rp, duong chu~n t6t co h~ 56 R=0,99997; R2=O,99993, M s6 tmmg quan rAt cM! D~ tinh dtrQ"cs6 ban copy Cl13 cac mflll nghien cUu cua gen GAPDH thea cong thlrc:
conc= 1 OA( -0.200xCT +10.386)
CT = -5.002Xlog( cone) +51.952
Trang 4f: y HQC VI~T NAM THANG 11 - 56 D~C BI~T/2014
~;
~ Gen DYS14 voi tin hieu huynh quang mall xanh diroi day ciing cho tin hieu t6t va duong
~-.:~".dnlendep voi ~~ s6 urong quan chat R=0.99988 va R~=0.99975
~.' Chung ta cothe tinh so ban copy cua gen DYS \ 4theo cong thuc sau:
cone= 10"lO.217"C1+10.647)
C1= -4.61 O:<log(conc) + 4~3.086
(
1-~ Cong thirc tinh s6 ban copy/I ml mall: _ Vpe! Ve:~. Trong do Q lit gia tri tinh duoc
~.d:~tich phan irng PC!t, Vext IIIth~ tich huy~t nrong d~ tach ADN phoi thai nr do
~~ 3.2 K~t qua chgy iljnb I.U'9ngcac 60 mfiu hUY~t.tuong phu nu' mang thai 0'cac tufin
~tiltlni' 6d~ntuAn 12 cua thai kyo
~" Ap dung quy trlnh tren d~ ti~~ ha~hdjnh Iu,?"g ki6m Iranbng do ADN ella thai nhi tro~g
F'. ;:~yet tuang nguai m~ tren 60 mau ba~g c~p moi va probe tren gen DYS 14, clmg v6'i 2 mall
~£:.,:'~ichUng am chung toi thu duQ'c cac ket qua sau:
~: Bang 1: Bang k~t qua s6 ban copy va n6ng et(l gen DYS 14 va n6ng d<)gen GAPDH a
~ de lUan 6 d~n 12 cua thaiky
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Bang qua Ii~u chay tren 60 phu ncr
mang thai tir tudn thrr 6 dSn tudn thu 12 cua
thai ky van6ng dQ ADN phoi thai Wdo trong
mau ngoai vi cua me a cac tu~n thai khac
nhau Co 30/60 twang hop mang thai nam
Tir bang k~t qua tren chung toi nh~n th~y
nong dQ ADN cua thai ky (1tirng nguaj khac
nhau la r~t khac nhau va co SI,l'bi~n thien tang
d~n n6ng dQ ham hrong cua ADN ph6i thai
tV do theo S\l' tang dau ella tu6i thai Tir bang
nay chung toi ciing th~y r~ng n6ng dQ trung
blnh ctta ADN phoi thai W do trong mau
ngo{li vi ctta mt(a cac tu~n ttr tuan thtr 6 d~n
tu~n thu 12 nay co dQ l~ch ehll:1n r~t 16n va
ian han ca ghi tri tru'1g blnh di~u nay chung
to n6ng dQ ADN phoi thai nay dao dQng (1
mQt khoang r~t Ion nhu atudn 6 ctta thai ky
n6ng dQ trung blnh ella ADN ph6i thai W do
trong mau ngo{li vi clta mt( la 50.69 ± 83.56
ban copy/mt hay a tll~n thir 12 la 206.63 ±
742,55 copy/mt Theo tac gia Lo YMD Tein
MSC, Lau TK va cQrtg s\T (1998) nghien cuu
tren 27 ph\! nil' mang thai aquy d~u etla thai
ky, qllY thtr 2 va qllY thu 3 Clla thai kyo cac
ph\l nil' nay mang tha~ b~ng ky thll~t IYF cho
thdy aquy 1 clla thai ky s6 han copy clla gen
oYS 14 trong hlly~t tuang Clla m~ vao
khoang 4,0 d~n 58,1 ban copy/ml di~u nay
ciing phu hqp voi nghien Ctru ella chung toi a
tudn thu 6 clla thai k5' m~c du (1cac tuan ti~p
theo nghien cuu ella chung toi s6 him copy co
cao han so voi tac gia nay Cung theo tac gia
Lo YMD Tein MSC Lau va S\1' (1 )
a giai doan muon quy thir 3 cua thai ky s6 ban copy cua gen DYS 14 trong huyet nrong dao dQngtir76.9 d~n 769 copy/ml
Nghien ciru cua Costa 1M Benachi A, Gautier E va cong str narn 2001 cung da dung ky thuat Realtime PCR d~ chimg minh r~ng ADN phoi thai W do t6n tai trong mall ngoai vi ella me Tac gia Alen Chan K.C va
cs (2004) tach chi~t va nghien CUlln6ng dQ
va ph an b6 kich thuoe ADN ph6i thai W do trong mall mt( cho thfty n6ng dQ ADN trong mall m~ chi~m 3.4-6,2% so voi ADN t6ng s6 tac gia nghien Ctru trtn 156 ph\! nti' mang thai ttr tll§n thu 7 d~n tu~n thtr 16 clla thai kY· Trong nghien cuu Clla chtmg t6i n6ng dQ ADN phoi thai a cac tll~n thai dao dQng tir
tu6i thai Chungwen Wei Devereux N Saller
va C9ngs\r (2001) du dinh ltrqng ADN tvdo ctla ph6i thai trong huy~t tucmg ella m~ etla
30 ph\! nfT mang thai ttT tll~n thiT 9 d~n tu~n thtr 17 Clla thai ky thdY co 19 trtT{'tng hqp dmmg Hnh vai gen DYS 14, s6 ban copy clla gen nay dao d9ng ttT 13.8 d~n 291 copy/ml, v6'i n6ng d() gen DYS14 nay trong hlly~t tuang la ttT 1.28 d~n49.7% voi 2 truang hQ'p n6ng d9 r~t cao la 29,2% bi hQi chtmg Down
va m9t tnrang hqp n6ng d9 cao d9t bi~n 47.69% nhung bQ nhi~m s~c th~ binh thuang 46XY
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Tir bang k~t qua so sanh tren cluing toi nhan
thay k~t qua ella cac tnrong hop nghien dill
tir tuan thir 8 d~n tufin thir 12 cua k9 thuat
Nested peR, Realtime peR sieu am tufin thir
16va sau sinh la phil hop nhau Voi oQchinh
xac la 100% va d9 nhay la 100% Voi tuan
thai 6 co 4/60 tnrong hop chay Realtime
-peR khong phu hop voi sieu am va sau sinh
do v~y dQ o~c hi~u Clla ky thu~t nay la 100%
va OQ nh\lY la 93,33% va 7/60 truang hqp
ch~y Nested peR khong phu hqp voi k~t qua
sieu am va sau sinh do v~y oQ d~c hi~u Clla
ky thu~t nay la 100% va oQ nh~y la 88,33%
3.4 K~t qua bi~n thisn nAng dQADN
phoi thai tl}' do trong mau ngo~i vi ciia me D~ kiern tra xem nong dQADN phoi thai
W do trong rnau ngoai vi cua me bien 06i theo tu~n thai ella thai nhi chung t6i tien hanh
dinh luong cac mftll nghien ciru nay a cac tuan tir 6 tufm o~n 22 tuan tuoi K~t qua cho thAy n6ng oQ bi~n thien nong dQ ADN phoi thai W do trong 1 ml huy~t tucmg co s\r thay obi gilra cac tuftn va n6ng oQ nay co xu huang tang len thea tu6i thai, m~c dll trong tlmg trtrang hqp n6ng dQ nay co hk giam nlmng sau do xu huang: tang tra l~i 6 cae tuan sall
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Hinb i Bieu do bien Ihiellnon~ dq AD:'i IIJ do ctia t1l1irn~ hqp :96 Hinh 9 BieD 110bien Ibien noug dQ AD:'i :\t do ctia lIuintg htrp 198
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Tir hinh anh cac bieu db v~ S\Tbien thien nong dQ ADN phoi thai chung toi nhan thay
nong dQ ADN phoi thai t\T do trong mau
ngoai vi ella my bi~n thien thea tung d6i
nrong va co tinh eh~t ca th~ Theo tac gia Lo YMD, Tein MSC, Lau TK va cong S\T(1998)
dll nghien cuu S\Tbien d6i ADN tv do trong huy~t tuong ella my ella 12 tnrong hop lam
thu tinh bng nghiem IVF a cac tu~n thai ttr tu~n d~u d~n 3 7 tu~n K~t qua cho thdYneng
dQ ADN ph6i thai bi~n d6i thee tung ca th~
va ding co xu huang tang sb ban copy khi tu6i thai cao d~n Tren bieu db bien thien
nbng dQ ADN tv do trong huy~t tuong cua
me a tren chung t6i nh~n thdY vai k~t qua tuang tvS\l' bi~n thien nay d~c tTUng cho tung
ca th~ va co xu huang tang len khi tu6i thai tang
IV KETLU~N
Trong nghien clru nay chung t6i da ph~\t hi~n va dinh lugng dugc nbng dQ ADN W do Clla thai nhi luu hanh trong huy~t tuang clla m~ b~ng phuang phap realtime peR Khao sat duQ'c nbng dQ ADN ph6i thai tvdo trong mau ngo~i vi cua m~acac tu~n tu6i thai khac nhau Nghien ClrU miy chi cho chung toi thAy duQ'c s\1'bi~n thien nbng dQ ADN ph6i thai tlJ
do a tirng ca th~ lil kh6ng gibng nhau Xu huang nbng dQ ADN phoi thai tv do t~ng dan
theo tu6i thai, thai diem tu6i thai thich hqp nhdt chung t6i IlJa chQn la 8 tu~n tu6i dthai diem tu6i thai nay nbng dQ ADN du 16n d~ co th~ tach dugc ADN phoi thai va co th~ (rng
TAl L1~UTHAM KHAo
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