DSpace at VNU: Phát hiện và định lượng ADN phôi thai tự do trong huyết tương người mẹ bằng phương pháp Realtime - PCR
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. s6 5.2014
TAP CHI Y • DlfOC HOC QUAN Slf
HAT HI~N VA £)INH LUQNG ADN PHOI THAI TlJ DO TRONG
TUONG NGUal Me BANG PHUONG PHAp REALTIME - peR
Tri~u
tu« Sang*;
Tr§n Van Khoa*; Dinh Doan Long**
phat hlen ra Sl,l'ton tal cua ADN phOi thai tl,l' do va te baa phOi thai tl,l' do trong huyet
ngU'cYime <1§ rna ra mot hU'ang ch~n doan trU'ac sinh mai bang bi~m phap khOng can thiep.
IU'Q'ngdU'Q'cADN phOi thai tl,l' do trong huyet tU'O'ngngU'cYime, chUng tOi sl) dvng phU'ong
- PCR dl,l'a tren ky thu~t TaqManPCR Realtime co sl) dvng dau do huynh Quang. Ky
cho phep quan sat dU'Q'Ccac tin hi$u huynh Quang cua gen dich va biet dU'Q'Cs6 ban copy
tCmg chu ky nnan gen. D6i tU'Q'ngnghien cuu: 40 thai phu dang mang thai tCPtuan thli
tuan thli 12 cua thai kyo Ket qua: gen GAPDH xu~t hi~n tin hi~u
40 mau voi 56 ban copy
tCP4,63E + 02 den 7,99E + 05/1 ml huyet tU'O'ng.Gen SRY xuat hi$n trn hi~u huynh Quang
mau thai nam tCP1,48E + 00 den 2,97E + 03. Hai mau noi chling am gen SRY khOng xuat
. tin hi$u va gen GAPDH xuat hi$n tin hi~u vai s6 ban copy mau noi chling am s6 1 la 1,33E +
so ban copy cua mau n¢i chling am 2 la 1,44E + 04.
a
a ca
TCPkhoa: ADN ph6i thai tl,l' do; Huyet tU'ong me; Djnh IU'Q'ng.
DETECTION AND QUANTITATION OF FETAL CELL DNA IN
MATERNAL PERIPHERAL BLOOD BY REALTIME - PCR
Introduction:
the discovery of the existence of fetal cells in maternal peripheral blood has opened
a new direction for early diagnosis of congenital anomalies by means of non-interference. To
fetal DNA circulating in maternal blood we used Realtime - peR method based on TaqMan
~·.::P£:R technique using fluorescent probes. This technique allows to observe the fluorescence signal
. the target gene DNA for us to know the number of copies of each target gene· at each time point
:·ci the peR process. Objects, chemicals DNA research methods: we shall select 40 pregnant
't.'Omenin the th week to the 1ih week of pregnancy. Along with two negative control sample of 2
DNA pregnant women not yet having sex once 5 ml of blood is taken DNA conduct split DNA
- separated plasma free plasma DNA as the other sample. Results: 40 samples were studied gene
'. GAPDH signals appear with number of copy of GAPDH gene in 1 ml is from 4.63E ~ 02 to 7.S9E +
05 in 1ml of plasma. SRY gene appearance Signals in the positive samples ADN the number of
copies of the SRY gene ranged from 1.48 to 2.97E + 03 in 1 ml of plasma. The form d08S net
appear SRY gene signal is the pregnant female gender, levels of GAPDH gene in pfasma DNA of
this sample ranged from 1.88 to 8.13E + 04E + 03 copies in 1 !Til of plasma For internal negative
control sample 2 signal SRY gene does not appear DNA GAPD.I-{gene signa! of internal negative
control sample 1 is 1.33E + 04 DNA internal negative controi 2 is 1.40E + 04 copies/mi.
* Key words: Fetal DNA; Realtime - peR: Non-interference .
• H9C vi{)n QUEmy
** £)9i h9c Y DllQ'C, £J?i h9c Qu6c gia Ha No;
NglPiYiphan hoi (Corresponding): Tri~u Tien Sang ()
Ngtw nh~n btli: 2810412014;Ngay phim bi~n danh gia btli bao: 1810512014
Ngay btli baa dlP9'cdang: 2610612014
45
-
-_-
_.-
~
-_
-_
-_
--::....,.,
------_._._----._-----
---
-
._-
-.~ ..--..----.
T~P CHI Y - DU'QC HQC QUAN S,! SO 5-2014
2 nCrgioi chua mang thai va chua quan
h$ tlnh due I~n nao, I~y 5 ml mau va tie!':
hanh tach huy~t tU'O'ng,tach ADN tl,l' 6
trong huy~t tU'O'ngnhu cac m~u nghie"
CLrUkhac. Nghien CLPUdU'Q'cthuc hien
tal Trung tam Nghien CLPU
Y DU'Q'choc
ouan Sl,l', Hoc vien Ouan y.
Nhi~u phuonq phap chlin doan truoc
sinh da dU'Q'cap dl,mg trong lam sang
nhu: sieu am thai, test sang loc bo ba
(triple test), la nhCrng bien phap khong
can thiep, nhU'ngcho k~t qua muon va do
d~c hieu th~p. Ngoai ra, con co cac phU'O'ng
2. H6a chit nghi€!n CLPu.
phap cho k~t qua sam nhu: choc hut rurcc
_ Hoa ch~t tach chi~t ADN: ethanol 70%,
6i, sinh thi~t gai rau. NhU'ngday la nhCfng
ethanol100%, proteinaseK, ruroc c~t, bOkit
phU'O'ngphap co can thiep, ty 1$ bi~n
Quiagen.
ch(J'ngcao cho ca thai nhi va san phu.
_ Hoa chat cho PCR: PCR Reaction
Vi$c phat hi~n ra 51,}'t6n tal cua t~ bao Mix, DNA Polymerase, Reverse primer,
ph6i thai trong mau ngo~i vi cua me da Forward primer, probe, nU'oc c~t khLi'ion
ra mot hLl'ang di mai cho ch~n doan va vo trung.
sam cac di t~t b~m sinh bang phLl'O'ng
_Thi~t bj: may I~c 6n nhi$t (Eppendori.
pMp khong can thi$p.
£)Lrc),may Iy tam cao t6c (Hettich, £)LPc).
ma
dinh ILI'Q'ngdLl'Q'cADN phoi thai ILI'u
hanhtrong mau me, chungtoi dung phLl'O'ng
phi3p realtime _ PCR dl,l'a tren ky thu~t
PCRTaqMan 5Li'dl,mgd~u do huynhquang.
Ky thu~t nay cho phep quan sat cac tin
tii~u huynh quang cua gen dich va cho ta
bit~t 56 ban copy cua tCfng gen dich
tll'ng thai dil!m cua qua trlnh PCR. Do v~y,
chung toi thl,l'c hi~n nghien c(J'u nay vai
ml,lCtieu: Phat hj{~n va ajnh iU'Q'ngADN
ph6i thai tl,f do trang huy§t tU'O'ngngU'&i
m{7 b~ng phU'ong phap realtime _ peR.
£)~
a
bu6ng thao tac PCR (My), may Realtime PCR (RotoGen, Quiagene), may PCR ASl
9700 (Applied BiosY5tem),tU hOt hoa ch~,
(Han Qu6c), may do n6ng do ADN (Nanc
Drop,My).
3. Phll'O'ng phap nghien CLPu.
Huy~t tU'O'ngsau khi Iy tam, lay 200 m)
tach chi~t bang bo kit cua Quiagen. ADN
sau khi tach chi~t dLl'Q'choa tan trorig 50 ui
H20 deian.
ADN nay sau do dLl'Q'ckhu€!chd~i bang
ky thu~t realtime _ PCR voi cac d~u do:
DOl TUdNG, HoA CHAT VA
SRY_F
5'-TGGCGATTAAGTCAAATTCGC·'j
PHUdNG PHAp NGHIEN c(1u
SRY_R
5'-CCCCCTAGTACCCTGACAATGTATT
.;J
ProbeSRY
5'-{F,AM)_TCTGCCTCCCTGACT-CCTCTACTGCT_(TAMRAj.?
GAPDH_F
5'-CCCCACACACATGCACTTACC.;J
GAPDH_R
5'-CCTAGTCCCAGGGCmGATT.;J
1. Dbi tll'Q'ng nghien CLPu.
40 pht,lnCrmang thai tll' tu~n th(J'7 d~n
tu~n th(f 12 cua thai kyoT~t ca dLl'Q'cI~y
5 ml mau to~m ph~n va Iy tam 5.000
vong/phut trong 5 phut r6i I~y huy~t
tLl'O'ng.Cung voi 2 m~u dbi chLrng am la
£)anh d§u d~u do bang 2 mau huynh
quang HEX va FAM.
46
~
.
-~
~ =-
_-
---
- ----=.
..
.
.
TAP CHi Y - DU'OC HOC QUAN SU'SO 5-2014
Thvc hien qua trlnh chay peR theo ehu trlnh sau:
..•Phan (rng peR v&i tbng th~ tich phan (rng 25 ul, trong do PCR master mix 10 ul;
SRY (F, R probe):(O,25:0,25:0,5) ul va primer GAPDH (F, R probe):(O,25:0,25:0,5ul);
8 ul.
10170%,
k be;> kit
95°C
10 phd!
95°C
1 phu!
60°C
1 phd!
19 s6 li~u: bang ph~n
gen da tieh hop san. va
..XI}
1 chu ky
55 chu
ky
mE§mcnuyen dl,mg ella may chay realtime-peR
cac thuat
toan
thong
ke tren
RotoGen -
phan rnem phan tich so Ii$u
Microsoft Office.
eaetion
primer,
KEr QUANGHIEN cuu
vA BAN LU';N
. 1. K~t qua sau khi chay dU'ang chu~n cua gen SRY va gen GAPDH trong m~u
uAn pha loang zy cac n6ng dQ 10\ 103,102,
endorf,
, D(re) ,
altime CRABI
)13ch~t
I (Nano
200 ml
1. ADN
9 50 ul
Ii bang
do:
Hinh 1: Hlnh anh phan tich dLl'cyngchuan cua gen GAPDH.
47
TI;P CHi y • Dt19C HQC QUAN 51!
sO 5-2014
Tin hi~u ella gen GAPDH len dep, dU'ong ehu~n t6t c6 h$ s6 R
= 0,99997;
R2::
0,99993, h~ s6 tU'O'ngquan rat chat. Tinh dU'Q'cs6 ban copy cua cac mau nghien
cua gen GAPDH theo cong
CUL
thuc:
cone= 10""(-0.200xCT + 10.386)
CT = -5.002 log{ cone 1 + 51.952
X
Gen SRY vai tin hleu huynh quang rnau xanh dU'ai day eOng eho tin hi$u
dU'ong chuan len dep vai h$ s6 tU'O'ngquan eh~t R
= 0,99988
va R2
tOt
va
= 0,99975.
"
lS
s,~dC",,",
",
cac-101.1121l'(T _.,O~71
CT .,.('6101og(concj t 49.086
Twe:Fbllilg
~:J~J
IIDPotted
Hinh 2: Hinh anh phan tie~ du'ong chu~n ella gen SRY.
Tinh s6 ban copy ella gen SRY thea eong thirc sau:
cone= 1 0 ......
(-0.21 7"CT
CT = -4.61 O"log(conc)
1 0.647]
+ 49.086
+
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TAP CHi Y - DlIOC HOC QUAN SlI SO 5-2014
29
297
8
2,87E + 02
38,21
1,08E + 04
30,27
2,6574
30
314
11
4,02E + 01
40,71
4,63E + 02
31,97
8,6825
31
Am n¢i
chling 1
1,33E + 04
29,08
0,00
32
Amn¢i
chling 2
1,40E + 04
27,75
0,00
33
270
8,4
8,13E+04
31,97
0,00
34
271
11
3,42E + 04
34,1
0,00
35
275
8
2,74E + 04
24,47
0,00
36
276
8
5,28E + 04
24,74
0,00
37
280
6,2
1.88E + 03
29,95
0,00
38
295
8
5,21E + 04
30,99
0,00
39
300
8,3
7,93E + 04
33,29
0,00
40
309
9
1,38E + 04
28,71
0,00
41
310
11,2
1,90E + 04
28,79
0,00
42
311
12
1AOE + 04
27,75
0,00
T~t ca 40 mau nghiEm CLrUgen GAPDH d~u xuat hien cac tin hieu v&i 56 ban copy
cua gen GAPDH nay/1 ml tLl' 4,63E + 02 d~n 7,99E + 05/1 ml huy~t tuonq. Gen SRY
xuat hi$n tin hieu & cac mau dU'O'ng tinh va 56 ban copy cua gen SRY dao dong tLl'
1,48 - 2,97E + 03/1 ml huy~t tU'O'ng, Cac mau kh6ng xuttt hi$n tin hleu cua gen SRY la
thai mang nhiem 5~C th~ gi&i tinh XX, nbng do ADN gen GAPDH huyM tU'O'ng cua
nhCPngmau nay dao dong tLl' 1,88E + 03 d~n 8,13E + 04 ban copy/1 ml huy~t tU'O'ng,
86i v&i 2 mau noi chLrng am, tin hleu gen SRY kh6ng xuat hi$n va tin hieu gen GAPDH
cua mau noi chLrng am 1 1131,33E + 04, noi chLrng am 2 1131,40E + 04 ban copy/ml.
KET LU~N
£)a
phat hi~n va dlnh 1U'Q'ngdU'Q'Cnbng
do ADN tl,l' do cua thai nhi luu hanh trong
huy~t tU'O'ngngU'ai me bang phU'O'ngphap
realtime - peR. Nghien CLrU nay mo ra
hU'&ng chan dean dl tat truce sinh va cac
b$nh di truy~n trLJ'&csinh bang bi$n phap
kh6ng can thi$p,
TAl L1~U THAM
KHAo
1, Bianchi OW, Flint AF, Pizzimenti MF,
Knoll JH, Latt SA. Isolation of fetal DNA from
nucleated erythrocytes in maternal blood, Proc
Natl Acad Sci USA 1990,87, pp,3279-3283,
50
2, Bianchi Ow, Shuber AP, DeMaria MA,
FougnerAC, Klinger KW Fetal cells in maternal
blood: determination of purity DNA yield by
quantitative peR. Am J Obstet GynecoL 1994,
171, pp.922-926.
3. Bianchi OW, Williams JM, Sullivan LM,
Hanson FW, Klinger KW, Shuber AP. peR
quantitation of fetal cells in maternal blood in
normal DNA aneuploid pregnancies. Am J
Hum Genet. 1997,61, pp.822-829.
4. Bianchi Ow, Zickwolf GK, Weil GJ,
Sylvester S, DeMaria MA. Male fetal progenitor
cells persist in maternal blood for as long as
27 years postpartum. Proc Natl Acad Sci. USA.
1996,93, pp.705-70S.
.
..
.
TAP CHi y. DlTOC HOC QUAN SlT 565.2014
BolADN CR. Setting microsatellites free.
2: 972-974CamaschellaC, AlfaranoA,
E, Travi M, Primignani P, Caligaris CF,
G (1990) Prenatal diagnosis of fetal
".",I,nrun Lepore-Bostondisease on maternal
blood.Blood. 1996,75, pp.2102-2106.
2,6574
8,6825
0,00
0,00
0,00
0,00
Ilite alterations in plasma DNA
all cell lung cancer patients. Nat Med.
2, pp.1033-1035.
0,00
0,00
0,00
Cheung MC, Goldberg JD, Kan YW.
diagnosis of sickle cell anemia DNA
by analysis of fetal cells in maternal
. Nat Genet. 1996, 14, pp.264-268.
0,00
0,00
0,00
8. Hamada H, Arinami T, Kubo T,
Hamaguchi H, Iwasaki H. Fetal nucleated
cells in maternal peripheral blood: frequency
DNA relationship to gestational age. Hum
Genet. 1993, 91, pp.427-432.
9. Heid CA, Stevens J, Livak KJ, Williams
PM. Realtime quantitative PCR. Genome Res.
1996,6, pp.986-994.
10. HolIADN PM, Abramson RD, Watson R,
GelfADN DH. Detection of specific polymerase
chain reaction product by utilizing the 5_-3_
exonuclease activity of the Thermus aquaticus
DNA polymerase. Proc Natl Acad Sci USA.
1991,88, pp.7276-7280.
),00
),00
ban copy
3en SRY
d¢ng tLl'
n SRY la
ang cua
!t tU'!>'ng.
GAPDH
rnl.
aria MA,
maternal
yield by
DI. 1994,
f
van LM,
p. peR
olood in
Am J
.:
'ell GJ,
)genitor
ong as
i. U~A.
51
ir
.-.
_---
- -
~_:f-=-~~
-_=-_ -_-- ~~~-~--
