Introduction: the discovery of the existence of fetal cells in maternal peripheral blood has openeda new direction for early diagnosis of congenital anomalies by means of non-interferenc
Trang 1Introduction: 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
:·cithe peR process Objects, chemicals DNA research methods: we shall select 40 pregnant 't.'Omenin the thweek to the 1ihweek of pregnancy Along with two negative control sample of 2
DNA pregnant women not yet having sex once 5ml of blood is taken DNA conduct split DNA
- separated plasma free plasma DNA asthe 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 2signal SRY gene does not appear DNA GAPD.I-{gene signa! of internal negative control sample 1is 1.33E + 04 DNA internal negative controi 2is 1.40E +04 copies/mi.
* Key words: Fetal DNA; Realtime - peR: Non-interference
TAP CHI Y • DlfOC HOC QUAN Slf s65.2014
HAT HI~N VA £)INH LUQNG ADN PHOI THAI TlJ DO TRONG
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§ rnara 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$u huynh Quang cua gen dich va biet dU'Q'Cs6 ban copy
atCmg chu ky nnan gen D6i tU'Q'ngnghiencuu:40 thai phu dang mang thai tCPtuan thli tuan thli 12 cua thai kyo Ket qua: gen GAPDH xu~t hi~n tin hi~u a ca40 mau voi 56 ban copy tCP4,63E + 02 den 7,99E +05/1 ml huyet tU'O'ng.Gen SRY xuat hi$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
TCPkhoa: ADN ph6i thai tl,l' do; Huyet tU'ong me; Djnh IU'Q'ng
DETECTION AND QUANTITATION OF FETAL CELL DNA IN
• H9C vi{)n QUEmy
**£)9i h9c Y DllQ'C, £J?i h9c Qu6c gia Ha No;
NglPiYiphan hoi (Corresponding): Tri~u Tien Sang (trieusangk83@yahoo.com)
Ngtw nh~n btli: 2810412014;Ngay phim bi~n danh gia btli bao: 1810512014
Ngay btli baa dlP9'cdang: 2610612014
45
- -_-
_.-~ -_
-_ -_._._ ._ - - - _- -.~ .
Trang 2T~P CHI Y - DU'QC HQC QUAN S,! SO 5-2014
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
canthiep, nhU'ngcho k~t qua muonva do
d~c hieu th~p Ngoai ra, con co cac phU'O'ng
phap cho k~t qua sam nhu: choc hut rurcc
6i, sinh thi~t gai rau NhU'ngday la nhCfng
phU'O'ngphap co can thiep, ty 1$ bi~n
ch(J'ngcao cho ca thai nhi va sanphu
Vi$c phat hi~n ra 51,}'t6n tal cua t~ bao
ph6i thai trong mau ngo~i vi cua me da
ma ra mot hLl'ang di mai cho ch~n doan
sam cac di t~t b~m sinh bang phLl'O'ng
pMp khong can thi$p
£)~ 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 a
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{~nva 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.
DOl TUdNG, HoA CHAT VA PHUdNG PHAp NGHIEN c(1u
1. Dbi tll'Q'ng nghien CLPu.
40 pht,l nCrmang 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
46
2 nCrgioi chua mang thai va chua quan h$ tlnh due I~nnao, I~y5 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 CLPUY DU'Q'choc
2 H6a chit nghi€!nCLPu.
_ Hoa ch~t tach chi~t ADN: ethanol 70%, ethanol100%, proteinaseK, ruroc c~t, bOkit Quiagen
_ Hoa chat cho PCR: PCR Reaction Mix, DNA Polymerase, Reverse primer, Forward primer, probe, nU'oc c~t khLi'ion
va vo trung
_Thi~t bj: may I~c 6n nhi$t (Eppendori
£)Lrc),may Iy tam cao t6c (Hettich, £)LPc) bu6ng thao tac PCR (My), may Realtime -PCR (RotoGen, Quiagene), may -PCR ASl
9700 (Applied BiosY5tem),tU hOt hoa ch~, (Han Qu6c), may do n6ng do ADN (Nanc Drop,My)
3. Phll'O'ng phap nghien CLPu.
Huy~t tU'O'ngsau khi Iy tam, lay 200m)
tach chi~t bang bo kit cua Quiagen ADN sau khi tach chi~t dLl'Q'choa tan trorig50 ui
H20deian
ADN nay sau do dLl'Q'ckhu€!chd~i bang
ky thu~t realtime _ PCR voi cac d~u do:
SRY_F 5'-TGGCGATTAAGTCAAATTCGC·'j
5'-CCCCCTAGTACCCTGACAATGTATT ;J SRY_R
ProbeSRY 5'-{F,AM)_TCTGCCTCCCTGACT-CCTCTACTGCT_(TAMRAj.? GAPDH_F 5'-CCCCACACACATGCACTTACC.;J
GAPDH_R 5'-CCTAGTCCCAGGGCmGATT.;J
£)anh d§u d~u do bang 2 mau huynh quang HEX va FAM
Trang 3k be;>kit
eaetion
primer,
<hLr ion
endorf,
, D(re) ,
altime
-CRABI
)13ch~t
I(Nano
200 ml
1.ADN
9 50 ul
Ii bang
do:
TAP CHi Y - DU'OC HOC QUAN SU'SO. 5-2014 Thvc hien qua trlnh chay peR theo ehu trlnh sau:
•Phan (rng peR v&i tbng th~ tich phan (rng 25 ul, trong do PCR master mix 10 ul; SRY (F, R probe):(O,25:0,25:0,5) ulva primer GAPDH (F, R probe):(O,25:0,25:0,5ul);
8ul
60°C
55 chuky
1 phd!
XI} 19 s6li~u:bang ph~n mE§mcnuyendl,mg ella may chay realtime-peR RotoGen -gen da tieh hop san va cac thuat toan thong ke tren phan rnem phan tich so Ii$u Microsoft Office
KEr QUANGHIENcuu 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 zycac n6ng dQ 10\ 103,102,
Hinh 1: Hlnh anh phan tich dLl'cyngchuan cua gen GAPDH
47
Trang 4Gen SRY vai tin hleu huynh quang rnau xanh dU'ai day eOng eho tin hi$u tOt va dU'ong chuan len dep vai h$ s6 tU'O'ngquan eh~t R=0,99988 va R2=0,99975
cone= 10""(-0.200xCT + 10.386)
CT = -5.002Xlog{ cone 1 + 51.952
TI;P CHi y • Dt19C HQC QUAN 51!sO5-2014
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 CUL
cua gen GAPDH theo cong thuc:
lS "
s,~dC",,", ", cac-101.1121l'(T _ ,O~71
CT ,.('6101og(concj t 49.086 Twe:Fbllilg
~:J~J
IIDPotted SdtinQl _!:.1
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 +1 0.647]
CT = -4.61 O"log(conc) + 49.086
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chling 1
32 chling 2Amn¢i
8 11
8,4 11 8 8 6,2 8 8,3 9 11,2 12
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~UTHAM 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,
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
50
Trang 7TAP CHi. y DlTOC HOC QUAN SlT.565.2014
BolADN CR Setting microsatellites free.
2: 972-974CamaschellaC, Alfarano A,
E, Travi M, Primignani P, Caligaris CF,
G (1990) Prenatal diagnosis of fetal
".",I,nrunLepore-Bostondisease on maternal blood.Blood 1996,75, pp.2102-2106
2,6574
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ban copy
3en SRY
d¢ng tLl'
ang cua
!t tU'!>'ng
GAPDH
rnl.
aria MA,
maternal
yield by
DI 1994,
van LM, f.:
p. peR
olood in
Am J
Ilite alterations in plasma DNA all cell lung cancer patients Nat Med
2, pp.1033-1035
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
'ell GJ,
)genitor
ong as
i U~A
i
r
-~_:f-=-~~
_=-_ -_ ~~~
-~-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
51