Real-time PCR

Cc vn c bn

Real-time PCR l g?

Trong k thut PCR, sau khi hon tt khuch i on DNA ch, ngi lm th
nghim phi tip tc lm mt s bc th nghim c kt qu xc nh c sn phm
khuch i mong mun trong ng phn ng hay khng, v giai on ny gi l giai on
th nghim sau PCR. Trong giai on ny, ngi lm th nghim c th thc hin in di
sn phm PCR trn gel agarose xem c vch sn phm khuch i ng kch thc
mong mun hay khng, cng c th thc hin th nghim lai vi cc on d c hiu
(trn mng, trn ging hay phin nha...) xem sn phm khuch i c trnh t mong
mun hay khng. K thut PCR m cn phi c giai on th nghim c v phn tch
sau khi hon tt phn ng khuch i, ngy hm nay c gi l PCR c in (classical
PCR).

Real-time PCR l k thut PCR m kt qu khuch i DNA ch hin th c ngay

sau mi chu k nhit ca phn ng, chnh v vy nn c gi l real-time; v do c
im ny nn vi real-time PCR ngi lm th nghim khng cn thit phi lm tip cc
th nghim c v phn tch kt qu xc nh c sn phm khuch i ch hay
khng v kt qu cui cng ca phn ng khuch i cng c hin th ngay sau khi
hon tt phn ng khuch i. Nh vy, nn c th ni real-time PCR l k thut nhn
bn DNA ch trong ng nghim thnh hng t bn sao da vo cc chu k nhit v kt
qu khuch i trong ng phn ng c hin th cng lc vi phn ng khuch i xy
ra ngi lm th nghim c th thy c.

Cc vn k thut c bn cn bit ca real-time PCR

1. Biu khuch i ca real-time PCR

Trong real-time PCR, hin th c bn ngi lm th nghim c th quan st

c trong qu trnh nhn bn DNA ca cc ng phn ng l mt biu khuch i
(amplification graph). Biu ny c trc tung (Y) l cng hunh quang pht ra t
cc ng phn ng khi nhn nh sng kch thch, cn trc honh (X) l cc chu k nhit.
Trn biu khuch i ny (biu 1), ngi lm th nghim s thy i vi tng

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ng phn ng, cng hunh quang m my ghi nhn c trong nhng chu k u
s rt thp v hu nh khng thay i, hin th bng mt ng thng nm ngang,
chng ta c th gi y l giai on hay giai on tim phc, v trong giai on
ny d DNA ch c th c nhn bn thnh cc bn sao nhng do s lng cha
gip cho cht pht hunh quang nhn c nh sng kch thch pht ra nh sng
hunh quang cng my ghi nhn. Nhng mt khi s lng bn sao ca DNA
ch t n mt ngng nht nh th nh sng hunh quang pht ra s cng
c my ghi nhn v lc ny chng ta s thy ng biu din khuch i bt u
ngc ln. Cng hunh quang trong ng phn ng t lc ny tr i s tng gp i
sau mi chu k nhit do s lng bn sao ca DNA ch tng gp i sau mi chu k.
Chng ta gi giai on ny l giai on ly tha v cng hunh quang, khng
phi l giai on tng trng ly tha v s lng bn sao ca DNA ch. Cng
hunh quang trong ng phn ng s tng trng n mt mc no th tng
trng s chm dn v t n bnh nguyn v cc bn sao ca DNA ch, do phn ng
cn dn dNTP v enzyme Taq polymerase khng hot ng hiu qu na, nn s
khng cn gia tng s lng theo cp s 2 na. Chng ta gi giai on ny l giai
on bnh nguyn.

Phn tch mt ng biu din khuch i (amplification curve) ca mt ng phn

ng sau khi hon tt c cc chu k nhit, chng ta s thy mt thng s ht sc quan
trng lun i km vi n, l chu k ngng (Ct, threshold cycle). Chu k ngng
hay Ct l chu k nhit m ti thi im ny thit b real-time ghi nhn c tn hiu
hunh quang pht ra t ng phn ng bt u vt qua cng hunh quang nn.
c th xc nh c cng hunh quang nn, thit b real-time thng ghi nhn
cng tn hiu hunh quang xut hin trong ng phn ng trong mt s chu k u,
chng ta gi l cc chu k nn (basal cycle), v ly trung bnh cng ca cc cng
hunh quang ny lm cng hunh quang nn. ng ct ngang i qua cng
hunh quang nn ny c gi l ng nn (base line). Chu k ngng l tr s c
xc nh bng s chu k m ng nn ct c ng bin din khuch i. Do
c tnh ton nh vy nn chu k ngng thng l mt s l (v d: Ct = 28.35) ch
t khi l mt s chn.

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Cng hunh quang

Giai on Giai on ly tha Giai on bnh nguyn

ng biu din
khuch i

Cng hunh quang nn

ng nn (base line)

5 10 15 20 25 30 35
Chu k nhit
Chu k nn
Chu k ngng (Ct)

Biu 1: Biu mt ng biu din khuch i ghi nhn cng hunh quang pht ra t ng phn ng khi
nhn c nh snh kch thch vo mi chu k nhit

C nhng ng phn ng c Ct sm v cng c nhng ng phn ng c Ct xut hin

mun hn, v cu hi c t ra l l do no lm c nh vy? Cu tr li chnh
xc l do s lng bn DNA ch ban u (starting quantity, Sq) c trong ng phn ng
nhiu hay t. Nu trong ng phn ng s lng DNA ch nhiu th s cn t chu k nhit
hn t n s lng bn sao ng phn ng cho c tn hiu hunh quang m
my s ghi nhn c, cn nu s lng DNA ch t hn th cn nhiu chu k nhit hn.

2. Biu chun ca real-time PCR

Nh ni Ct ca mt ng phn ng real-time PCR sm hay mun (Ct thp hay

Ct cao) l ty thuc vo s lng bn DNA ch ban u c trong ng phn ng. y
chnh l mt c im vt tri ca real-time PCR so vi PCR c in, v nh da vo
c im ny m ngi lm th nghim xc nh c s copies ca tc nhn ch c
trong mu th, mt thng s m PCR c in khng th no lm c c kt qu
chnh xc. Ngi lm th nghim ch c kt qu PCR c in sau khi hon tt phn
ng khuch i, v kt qu ny nu nh lng c th cng ch l kt qu nh lng

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s bn sao ca DNA ch
ng biu din khuch
i ca cc mu chun
sau khi hon tt khuch
i. M trong PCR, s
lng bn sao cui cng ng biu din khuch
i ca cc mu th
khng phn nh c mt
cch chnh xc s lng
bn DNA ch ban u c
trong mu th v trong a
s cc trng hp s Biu 2: Biu khuch i v ln cc ng biu din khuch i
ca cc mu th v cc mu chun
lng bn sao c trong
ng phn ng PCR l s lng cc i sau khi PCR t c giai on bnh nguyn.

Tuy nhin real-time PCR xc nh c chnh xc s lng bn ch ban u

c trong mu th, ngi lm th nghim phi thc hin real-time PCR cc mu th
cha cc s lng bn DNA ch ban u cn xc nh s lng cng lc vi cc mu
chun cha s lng DNA ch ban u bit r s lng, v thng th cc mu
chun l cc mu pha long theo h s pha long 10 cha s lng DNA ch ban u.
Sau khi hon tt cc chu k nhit ca real-time PCR, trn biu khuch i (biu
2), cc mu chun v cc mu th s c cc ng biu din khuch i ca n v
tng ng vi cc ng biu din
khuch i ny, cc mu chun v
mu th u c mt chu k ngng
(Ct).

ng biu din v ln mi
quan h gia chu k ngng vi s
lng bn DNA ch ban u c
trong cc mu chun c gi l
ng biu din chun (standard
curve). y l mt ng thng
Biu 3: Biu chun v ln ng biu din chun v mi
tuyn tnh i qua cc im ta quan h gia s lng bn DNA ch c trong cc
mu chun v chu k ngng tng ng. Trong
xc nh bi s lng bn DNA biu trn ny, chng ta E2-E4-E7 l cc ng
phn ng cha cc mu chun cn B4 l ng phn
ch ban u ca tng mu chun ng cha mu th.

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(trn trc tung) v chu k ngng ca ng phn ng cha s lng bn DNA ch ny
(biu 3). Trn biu chun ny, trc tung (Y) l Ct cn trc honh (X) l s lng
bn DNA ch ban u c trong cc mu chun. Do cc mu chun thng c pha
cch nhau theo h s pha long 10, nn s lng bn DNA ch trong cc mu chun
c biu th bng logarith c s 10 (log10) ca s lng ny. Do vy tr 1.5, 2, 2.5, 3,
3.5, 4, 4.5 trn trc X l tng ng vi cc s lng bn ch l 101.5, 102, 102.5, 103,
103.5, 104, 104.5 tc l 32, 100, 316, 1000, 3162, 10000, 31623 copies trong ng phn
ng.

C hai thng s c my tnh ton v hin th trn mt ng biu din chun.

Hai thng s ny rt c gi tr ngi lm th nghim c th nh gi c thao tc
pipetting ca mnh trong qu trnh lm th nghim.

Thng s u tin l h s tng quan R2, nh gi chnh xc ca thao tc

pipetting ly c ng th tch mong mun hay khng. Tr s R2 phi t l trn hay
bng () 0.99 c ngha l ng biu din chun phi t tuyn tnh cao. Khi ngi
lm th nghim khng ly c cc th tch chnh xc th chc chn cc th tch mu
chun cho vo real-time PCR mix s khng th no chnh xc c, nn R2 chc chn
s khng th no t 0.99.

Mt tr s na rt c gi tr m ngi lm th nghim phi bit r l hiu qu

PCR, c gi l E% (PCR efficiency). Mt khi PCR t hiu qu l tng, th c sau
mi chu k nhit cng hunh quang trong mt ng phn ng phi tng gp i,
cn nu hai ng phn ng c s lng bn DNA ch ban u cch nhau mt h s
pha long A, th hai ng biu din khuch i s cch nhau n chu k vi cng
hunh quang ca hai ng phn ng cng s cch nhau mt h s pha long A = 2n. Nh
vy, nu pha long l 10 ln th Ct ca cc pha long DNA ch ca mu chun
lin tip nhau s cch nhau l 3.32 chu k (tnh ton t cng thc trn vi A = 10, s
tnh c n = 3.32 chu k). Trong biu chun vi cc mu chun cch nhau qua
pha long h s 10 th hiu qu khuch i E c tnh l bng cng thc 10-1/slope vi
slope chnh l c ca ng biu din chun. Mt cch l tng, hiu qu khuch
i E ca PCR phi t c l 2 ngha l s lng bn sao nhn bn t bn DNA ch
phi tng gp i sau mi chu k nhit trong giai on tng trng ly tha, tc l 2 =
10-1/slope. T cng thc ny, chng ta s tnh ra c ng biu din chun l tng

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phi c c (slope) l -3.32. Nh vy, chng ta thy dc chnh l s cch bit
nhau v chu k ngng gia cc ng chun c s lng gim dn theo h s pha long
10. Hiu qu khuch i cng c th tnh bng phn trm (%) vi cng thc tnh t E
l: E% = (E-1) x 100%. Vi E t l tng l 2 th E% = 100 %. i vi bt c mt
ng biu din chun no, u c th tnh c hiu qu khuch i E % = (10-1/slope
1) x 100 %. Do vy nu dc ca mt ng biu din chun l -3.436 th E s l
10-(1/-3.436) = 1.954, vy th hiu qu % ca khuch i (hiu qu PCR) s l: E % =
(1.954 1) x 100% = 95.4 %, c ngha l trong giai on tng trng ly tha s lng
bn sao sau mi chu k nhit s c nhn ln 1.954 ln hay hiu qu PCR l 95.4 %.
D nhin hiu qu PCR l tng l 100 %, nhng trong thc t th hiu qu PCR chp
nhn c phi trong khong 90 % - 105 %. Ngi lm th nghim phi bit dng hiu
qu PCR hin th trong biu chun nh gi thao tc pipetting pha long mu
ca mnh. Sai lm u tin c th gp trong thao tc pha long mu l mang lng
DNA ch t mu nng cao xung mu c nng thp hn, m thng l do
khng thay u pipette sau khi mang th tch t nng cao xung nng thp, v
vy m s lng bn DNA ch c trong cc mu chun c pha long s nhiu hn
tnh ton. V d nu thao tc pha long chnh xc, th t 10.000 copies xung 1.000
copies ri 100 copies s chnh xc nh vy; nhng nu khng thay u pipette sau mi
ln ht mu v lng bn ch mang theo u pipette l 40% th chng ta s c cc
s lng s l 10.000 copies xung 4.000 copies ri 1.600 copies. V vy nn hiu qu
PCR c tnh ton bng gi tr E s khng t c l tng 90 % n 105 % m s
cao hn 105 %. L do l chu k ngng ca cc mu chun s b gn nhau hn lm
cho dc ca ng biu din chun thp i. Ngc li nu thao tc pha long m
khng cho th tch mu chun ht t mu c s lng DNA chun cao pha mu
chun lng thp hn (sai lm ny c th gp khi dng cc u pipette b dnh dung
dch trn thnh nn khng y c ht lng mu t u pipette xung mu k), v
chnh nh vy nn chu k ngng ca cc mu chun lin tip nhau s cch xa nhau
lm cho dc ca ng biu din chun thp i, v hiu qu PCR s di 95 %.
Hiu qu PCR cng l mt thng s rt c gi tr nh nghin cu ti u c k
thut real-time PCR m mnh. Nu hiu qu PCR thp th nguyn do c th l thit k
mi cha t nhy, do tch chit DNA ch cn cc c ch ngn cn phn ng

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khuch i. Nu hiu qu PCR cao th nguyn do li c th l do s bt cp khng c
hiu ca mi hay ca on d.

Nh vy, vic thit lp biu chun l rt cn thit khi lm real-time PCR chn
on, c bit khi xc nh chnh xc s lng tc nhn ch ban u c trong mu
th. Biu chun c cng dng trc tin l nh gi c thao tc pipetting ca
ngi lm th nghim c t khng. y l mt vic l ht sc quan trng v nu thao
tc pipetting m khng chnh xc th s khng th no c c kt qu nh lng
chnh xc c. Do vy s tht l sai lm v thiu hiu bit nu nh sn xut b thuc
th real-time PCR dnh nh lng tc nhn ch li cung cp sn cc PCR mix c
cho trc cc lng chun ca DNA ch, gi l cc b chun, m khng cho ngi
lm th nghim t pha long cc mu chun cho vo real-time PCR mix.

Sau khi nh gi c thao tc pipetting ca ngi lm th nghim l t,

ngi lm th nghim s s dng cng dng th hai ca biu chun, l xc nh
c s lng tc nhn ch ban u c trong mu th. Gi tr ny c tnh ton nh
vo hm s biu th mi tng quan gia chu k ngng (Y = Ct) vi log10 ca s
lng bn DNA ch ban u c trong ng phn ng (X = log10 Sq). Hm s l: Y =
[slope (X)] + intercept, v cc thng s slope v intercept u hin th trn biu
chun. T hm s ny, ngi lm th nghim s hiu c ti sao my tnh hin th
c Sq, l nh tnh ton t Sq = 10[(Ct intercept)/slope]. T Sq ny, ngi lm th
nghim s tnh c s lng tc nhn ch ban u c trong mu th da vo hiu qu
tch chit nucleic acid ch t mu th v cc pha long nu c trong qu trnh tch
chit cng nh thc hin real-time PCR. V d: khi s dng cc b thuc th pht hin
v nh lng HCV do cng ty Nam Khoa sn xut th s copies HCV c trong 1 ml
huyt thanh th nghim s bng N x 71 vi N l s copies nh lng c trong ng
phn ng, hay khi khi s dng cc b thuc th pht hin v nh lng HBV do cng
ty Nam Khoa sn xut th s copies HBV c trong 1 ml huyt thanh th nghim s
bng N x 50 vi N l s copies nh lng c trong ng phn ng (xem ph lc
bit cch tnh ton c cc h s ny).

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My real-time PCR

iu kin u tin c th thc hin c real-time PCR l phi c my real-time

PCR. My ny cng c mt bung nhit chy c chng trnh lun nhit nh my
PCR bnh thng, nhng c thm mt thit b c gi l thit b real-time. y l mt
thit b quang hc c hai chc nng: (1) C cc ngun sng pht ra c cc tia sng
kch thch (excitation light) c bc sng xc nh ln cc tube phn ng real-time PCR;
(2) C c camera hay cm bin quang ghi nhn c nh sng hunh quang pht ra
(emission light) t cc tube phn ng real-time PCR khi cc tube phn ng ny c
chiu cc tia sng kch thch.

Ty theo hng v model sn xut m c nhiu kiu thit b real-time khc nhau. Cc
kiu thit b ny khc nhau v cch pht ra ngun sng kch thch v cch ghi nhn c
nh sng hunh quang c pht ra t cc tube phn ng. Tm tt c cc kiu sau y:

1. S dng ngun sng kch thch l n tungstene v dng cc knh lc chiu

ngun sng c di sng nht nh ln ton b cc ging cha tube phn ng

Hnh 20 minh ha s
a mang v thay i v tr
cc knh lc nh sng
mt thit b real-time dng hunh quang n CCD
camera

n tungstene lm ngun sng Gng phn chiu ngun sng

kch thch nhng cho php nh
CCD
Camera

sng hunh quang pht ra i qua

kch thch. Ngun sng ny i
qua mt knh lc mu c ng i ca nh sng
Cc knh lc nh sng
hunh quang pht ra

ngi s dng chn (bng

chng trnh iu khin Ngun nh sng kch
thch l n Tungsten

xoay a chn knh lc mu Cc knh lc ngun

nh sng kch thch

thch hp n ng v tr) ch Bung nhit

ca my PCR
cho mt loi nh sng c
a mang v thay i v tr

di sng thch hp i qua. cc knh lc ngun sng

kch thch

nh sng kch thch c di Hnh 20: Minh ha s mt thit b real-time dng n tungsten lm
ngun sng kch thch v CCD camera ghi nhn ton b tn
sng chn lc ny c hiu hunh quang ca cc tube phn ng b chiu sng bi
ngun sng kch thch
gng dichroic phn chiu
xung bung nhit c cc tube phn ng. Trong tube phn ng c cha mt loi ha
cht c kh nng pht hunh quang, nu c s hin din ca si i DNA c khuch

41
i t DNA ch v b chiu sng bi nh sng kch thch c di sng thch hp.
nh sng hunh quang pht ra t tube phn ng s i qua gng dichroic, qua knh lc
mu hunh quang c ghi nhn bi mt CCD camrea. CCD camera ny s chp
hnh nguyn bung nhit ca my PCR ghi nhn tn hiu v cng hunh
quang c pht ra t cc tube phn ng qua mi chu k nhit v a v b vi x l
ca my vi tnh ri hin th real-time ln mn hnh bng biu ngi lm th
nghim thy c cng ca tn hiu hunh quang pht ra t mi ging phn ng
qua cc chu k nhit. Thit b real-time kiu ny c th thy c trong cc my real-
time PCR th h IQ (v d IQ, MyIQ, IQ5) ca hng Biorad.

2. Thit b real-time dng si quang hc (fiber optic cables) a ngun sng

kch thch n cc tube phn ng

Trong thit b ny, ngun sng kch thch c th l n laser, hay n tungstene,
c cc si quang hc dn trc tip n cc tube phn ng, v cc si quang hc hc
ng thi
cng lm Ngun sng laser
hay tungstene
Bung nhit

lun nhim Fiber optic cable

v dn nh
Fiber optic cable

sng hunh Thu knh nh

Tube phn ng
quang pht
ra n CCD Bung nhit

camera.
Thit b real- Hnh 21: Minh ha mt thit b real-time dng si quang hc a nh sng kch thch
n tube phn ng
time kiu
ny c th c thy trong cc my realtime PCR ca hng ABI, nh cc my real-
time ABI 7000 hay ABI 7.500 (hnh 21).

3. Thit b real-time dng n led lm ngun sng kch thch

Trong thit b ny, ngun sng kch thch c s dng l cc n LED c gn

trn mt gi v gi ny di chuyn st trn bung nhit (hnh 22) chiu nh snh
kch thch ln cc tube phn ng v nhn nh sng hunh quang pht ra (my real-time
PCR model CFX 96 ca Biorad), hay l c c nh ti mt v tr trong bung nhit
v cc tube phn ng c di chuyn n v tr ny nh cc tube phn ng c gn
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 trn mt gi xoay trn (my lun nhit
Ligh-cycler ca Roche hay Rotor Gene
ca Corbett Research). Li im ca thit
b real-time ny l i sng ca n LED n LED

c th ko di rt lu, c th n hng Lc bc sng

kch thich

chc ngn gi.

Ha cht v thuc th cho real-time PCR Cm bin quang

Gng dichroic

Cha kha k thut chnh ca ha cht v

Tube phn ng
thuc th c trong tube phn ng real-time
Bung nhit
PCR chnh l cht hunh quang c thm
vo PCR mix. Cht hunh quang ny phi
Hnh 22: H thng 6 n LED cng vi 6 cm bin
lm th no tube phn ng c th pht quang c gn trn mt gi v c di
chuyn st trn bung nhit c trong thit
c hunh quang khi b chiu bi ngun b Real-time PCR ca my real-time PCR
CFX 96 ca Biorad
sng kch thch mt khi trong tube phn ng
ny c s hin din sn phm khuch i ca PCR t DNA ch, v s khng th pht
c hunh quang nu khng c sn phm khuch i trong ng. Cho n nay, cc nh
khoa hc tm ra c nhiu cht hunh quang nh vy. Trong phm vi ni dung ca
bi ny, chng ti ch xin trnh by mt s cht hunh quang thng c s dng nht.

1. Cht pht hunh quang l mt loi mu hunh quang chn vo si i DNA

Mu hunh quang chn vo si i DNA l mt loi mu hunh quang c i lc

rt cao khi c s hin din ca si i DNA v i lc ny l do kh nng chn ca mu
vo gia si i DNA v lm cho si i DNA pht c nh sng hunh quang khi
nhn c ngun sng kch thch.

Nguyn tc k thut real-time PCR dng mu hunh quang chn

Nguyn tc ca k thut ny l khi khng c s hin din sn phm khuch i

ca PCR, cht hunh quang b phn tn trong dung dch PCR mix, do vy m tube
phn ng s khng b pht hunh quang hay ch pht hunh quang khng ng k khi
b chiu bi ngun sng kch thch. Nhng khi c s hin din sn phm khuch i
ca PCR th mu hunh quang s b chn vo v tp trung trn cc si i DNA ca
sn phm khuch i v s lm cho tube phn ng b pht hunh quang khi b chiu bi

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ngun sng kch thch. Ging nh trn phi c nhn xung mt bin vo ban m m
trn mt bin c ri rc cc thuyn con sng n. Nu cc thuyn con ny cch xa nhau
th chng ta s thy mt bin ti en, nhng nu chng tp trung li vi nhau th chng
ta s thy trn mt bin c nhng vng sng do tp trung nh sng t cc thuyn.

Khi thy ethidium bromide c s dng cho nguyn tc real-time PCR ny,
nhng sau ny cc nh khoa hc s dng SYBR I v cc u im vt tri hn nh
mu hunh quang nn rt thp, kh nng chn vo si i cao nhng khng lm cho
si i b gn cht vo nhau khi b bin tnh nh vy m nh hng t ln hiu qu
PCR. Hnh 23 minh ha nguyn tc hot ng ca real-time PCR dng mu chn l
SYBR I lm cht pht hunh quang. Ph quang ca SYBR l: l = 488 cho ngun sng
kch thch v l = 522 cho hunh quang pht ra.

Thit k mi cho real-time PCR dng mu hunh quang chn

Thit k mi cho real-time PCR dng

Ngun sng nh sng
kch thch hynh quang
mu hunh quang chn cng theo cc qui
lut thit k mi chung cho PCR, l: (1)
trnh hin tng chn tc u 5 hay 3;
(2) trnh bt cp gia primer vi nhau hay
cng primer vi nhau, nht l ngay u
3; (3) Trnh bt cp khng c hiu trn
trnh t ch; (4) thnh phn GC trong
khong 40-60%; (5) Trong trnh t mi, (1) (2)

trnh lp lin tip trn 3 base G hay C; (6)

Nn thit k u 3 c base l G hay C
trnh sn phm khuch i khng c
hiu; (7) Nn thit k nhit bt cp
ti ho l t 55oC n 65oC khng b
khuch i khng c hiu v nhit bt
Hnh 23: C ch hot ng ca SYBR I (1) Khi
cha c sn phm khuch i th ng th
nghim khng pht c hynh quang khi
nhn c nh sng kch thch, (2)
Nhng khi c hin din ca sn phm
khuch i th SYBR I chn vo v tp
trung trn phn t DNA , lm cho ng
phn ng b pht hunh quang khi nhn
c nh sng kch thch

cp qu thp. Ngoi ra, i vi real-time

PCR th lu thm mt yu t k thut na, l sn phm khuch i: nn thit k
mi c sn phm khuch i t 75 n 200 bps. Khng nn t hn 75 bps v nh vy
s kh phn bit c sn phm khuch i c hiu vi sn phm khuch i ca
44
 dimer-primer (xem phn phn tch melt curve), khng nn di qu 200 bps v nh vy
s kh t c hiu qu PCR l tng v nh vy th biu chun s khng t
c th cho c kt qu nh lng chnh xc (xem phn biu chun ca real-time
PCR).

Cch pha real-time PCR mix s dng mu hunh quang chn l SYBR I

thc hin c k thut real-time PCR s dng SYBR I lm cht pht hunh
quang th ngi lm th nghim phi pha PCR mix trong c thm SYBR I vi nng
1X (pha t stock 10.000X), nng MgCl2 n 3mM, v thm mt t DMSO,
BSA... Ngoi ra, ty thuc vo thit b real-time PCR c i hi PCR mix phi thm
mu hunh quanh nn hay khng thit b nhn din c v tr v xc nh c gi
tr ngng hunh quang ban u ca tng ging th nghim, m ngi lm th nghim
c cho thm cht hunh quang nn vo PCR mix hay khng. V d vi cc my real-
time th h IQ ca Biorad th hunh quang nn cho vo PCR mix l fluorescent, cn
vi cc my ABI 7000 th hunh quang nn l ROX. c th thc hin pha real-time
PCR mix mt cch n gin m li hot ng mt cch hiu qu, ngi lm th nghim
c th pha t real-time PCR master mix do cc hng sn xut c uy tn cung cp c
cha sn SYBR I, enzyme Taq polymerase, dNTP v MgCl2. Vi PCR master mix gi
l SYBR PCR master mix ny, ngi lm th nghim ch cho thm mi, nng 10-25
pm cho mt th tch phn ng, v nu cn th cho thm dUTP v UNG chng ngoi
nhim, l c c real-time PCR mix thc hin real-time PCR. Bng 4 di y

NK
Bng 4: Cch pha SYBR PCR master mix t SYBR PCR master mix 2X sau phn thnh 100 mix vi th
tch phn ng l 25 ml v th tch mu cho vo PCR mix l 5 ml.

STT Thnh phn gc Nng hay Nng hay hm lng Nng hay hm lng Th tch phi ly
hm lng gc trong 1 th tch p trong 100 th tch phn ng pha master mix

NK
1 SYBR PCR master mix 2X* 2X 1X 1X 1250ml

5 Mi xui 100pm/ml 10pm 1000pm 10ml

6 Mi ngc 100pm/ml 10pm 1000pm 10ml

7 dUTP 100mM 200mM 200mM 5ml**

8 UNG 1U/ml 1U 100U 100ml

9 Nc kh ion Cho (qsp) 20ml Cho (qsp) 2000ml 625ml

NK
* SYBR PCR master mix 2X c cung cp c sn MgCl2 6 mM. **c tnh ton theo cng thc CV = CV, vi C l nng gc,
V l th tch gc cn phi ly, C l nng cn phi pha, v V l tng th tch phn ng (trong v d ny l 2500 ml, tng th tch
ca 100 phn ng, mi phn ng 25 ml).

45
 NK
trnh by cch pha mt SYBR PCR master mix cho 100 mix t SYBR PCR master
mix 2X do cng ty Nam Khoa sn xut.

Chng trnh lun nhit real-time PCR dng SYBR I l mu chn

Vi real-time PCR s dng SYBR I lm cht pht hunh quang; thit b real-time
s ghi nhn c s pht hunh quang ti a t ng phn ng, khi nhn nh sng kch
thch, l cui giai on nhit ko di ca chu k PCR. Do vy mt chng trnh
lun nhit cho real-time PCR s dng SYBR c th l: 1 chu k: 40oC/10 (nu trong
PCR mix c dUTP v UNG), 1 chu k: 95oC/5 (nu dng hot-start Taq polymerase1);
35-40 chu k, l chu k lun nhit cho PCR gm 3 giai on nhit : 94oC/15-30,
55oC - 65oC/30-60, 72oC/30-60. Trong cc chu k lun nhit PCR ny, chn lnh
thit b real-time pht ra ngun sng kch thch v ghi nhn nh sng hunh quang pht
ra t ng phn ng trong giai on nhit bt cp 55oC - 65oC, my s t ng thc
hin hai chc nng quang hc quan trng ny giai on cui ca bc nhit bt
cp.

Chng trnh phn tch nhit chy

V SYBR I l mu chn cho bt c si i DNA no xut hin trong ng phn ng

sau cc chu k nhit k c cc si i DNA khng
phi khuch i c hiu t DNA ch, do vy s xut 3
5 [1]
5
hin ng biu din khuch i trong ng phn ng 3

s khng c hiu 100 % cho s c mt ca sn phm 3

5
[2]
khuch i c hiu t DNA ch. Nh vy th trong 5
3
ng phn ng, sn phm khuch i no thng c th
xut hin m khng phi l sn phm khuch i t
DNA ch? chnh l cc sn phm khuch i t Hnh 24:
Polymerase khi cn hot ng hu
cc dimer primer, thng xy ra vo cc giai on hiu th s khng th ko di mi
ca dimer primer t u 3 [1],
cui ca cc chu k nhit, khi m enzyme Taq nhng trong cc giai an cui ca
chu k nhit, polymerase khng cn
polymerase hot ng khng cn hiu qu v c hiu hu hiu na nn s ko di c
mi t u 3 to c sn phm
na. Dimer primer l s bt cp ln nhau gia mi do khuch i ca dimer primer [2]

1
Hot start Taq polymerase l Taq polymerase m nhit thng b bt hot v c khng th c hiu (hay ha cht) bm ln
o
im hot ng ca enzyme. Khi b un ln 95 C trong 5 pht th khng th dc hiu s b pht hy, Enzyme s hot ng. Hot start
Taq polymerase thng c s dng trnh cc bt cp khng c hiu nhit di nhit bt cp c hiu ca mi

46
c nhng v tr trn trnh t mi mang nhng base b sung c vi nhau. Tuy nhin
trong cc chu k u khi m enzyme polymerase cn hot ng hiu qu th s khng
to c sn phm khuch i c hiu do s ko di ca hai mi khi chng bt cp
ln nhau ch vi v tr nucleotide m khng bt cp c u 3 (hnh 24 [1]). Tuy
nhin trong cc giai on cui ca PCR, enzyme polymerase tr nn hot ng km
hu hiu nn n vn c th ko di c cc mi khi chng bt cp ln nhau d u
3 ca mi vn khng c bt cp t hiu vi nhau, chnh v vy nn mi to c cc
sn phm khuch i khng c hiu t cc dimer primer (hnh 24 [2]).

Sn phm khuch i t dimer primer s khc bit vi sn phm khuch i t

DNA ch chnh l chiu di, nu t dimer primer th chiu di s khng bao gi
vt qu 50 55 bps, cn nu t DNA ch th chiu di thng qu 100 bps. Nh vy,
c th phn bit c c ng biu din khuch i ca ng phn ng l ca sn
phm khuch i c hiu t bn DNA ch hay l t dimer primer, phi da vo mt
tnh cht c trng gip phn bit c chiu di ca hai loi sn phm khuch i ny,
v tnh cht c trng chnh l nhit chy (melting To, Tm), l nhit lm cho
si i DNA b bin tnh 50 % tc l c 50 % s si i b bin tnh hon ton. Nhit
chy ca si i DNA ty thuc vo thnh phn GC v chiu di ca n: Thnh
phn GC cng cao th Tm cng cao, si i cng di th Tm cng cao. Sn phm
khuch i t DNA ch di hn nn s c Tm cao hn l Tm ca sn phm khuch i
t dimer primer. bit c Tm ca sn phm khuch i c trong ng phn ng, sau
khi hon tt cc chu k lun nhit ca real-time PCR, ngi lm th nghim s tip tc

Biu 4: Biu [1] l biu chy (melt curve chart) c my hin th real-time trong sut qua trnh thc
hin chng trnh lun nhit melt-curve, phn tch trn biu ny chng ta s thy ng phn ng
o
c Tm thp 78 C l ng c sn phm khuch i l t dimer primer, cn cc ng phn ng c Tm
o
cao n 85 C l tht s dng tnh cc sn phm khuch i t DNA ch. Biu [2] l biu
nh chy (melt curve peak chart) cho php xc nh Tm d dng v chnh xc hn nh cc nh biu
chy ca tng ng phn ng

47
cho my chy thm mt chng trnh phn tch nhit chy, gi l chng trnh
melt-curve. Ty thuc vo tng loi my real-time PCR m ngi lm th nghim s
dng chng trnh melt-curve c ci sn trong my, hay l t to chng trnh melt
curve m mnh thy thch hp hn. i vi cc my real-time PCR th h iQ ca
Biorad th chng trnh melt-curve l, trc ht bung nhit s a nhit ln 95oC
trong 1 pht lm bin tnh hon ton cc sn phm khuch i c trong ng phn
ng, ri h xung nhit 55oC trong 1 pht tt c cc sn phm khuch i ny bt
cp hon ton thnh si i. Sau thc hin chng trnh melt-curve a nhit
bung nhit t 55oC ln 95oC trong 80 bc tng nhit , mi bc tng 0.5oC v
gi trong 10 giy; ng thi trong mi bc ny, thit b real-time cng s chiu ngun
sng kch thch v ghi nhn v hin th trn th cng nh sng hunh quang pht
ra t cc ng phn ng ln mt biu chy (melt curve chart). Phn tch trn biu
hin th real-time ny, chng ta s thy i vi tng ng phn ng th cng hunh
quang pht ra s gim dn theo cc bc tng nhit trong bung nhit, l do l cc
sn phm khuch i c trong ng phn ng s b bin tnh dn dn theo s gia tng
nhit trong bung nhit. n mt bc tng nhit m trng khp vi Tm
ca sn phm khuch i trong ng phn ng th cng hunh quang s gim t
ngt v c n 50 % sn phm khuch i ny b bin tnh thnh si n cng mt lc,
do vy bc nhit ny chng ta s thy ng biu din cng hunh quang
pht ra t ng phn ng khng cn l mt ng thng i xung u nh trc m b
chi xung thp hn nhiu so vi dc ca n. Nh vy m ngi lm th nghim
khi quan st din tin ca qu trnh v real-time biu chy, s hon ton c th c
th xc nh c Tm ca sn phm khuch i c trong ng phn ng (biu 4 [1]).
gip xc nh c d dng hn Tm, sau khi hon tt chng trnh melt-curve, my
real-time PCR s thay i hin th biu chy thnh mt khc gi l biu nh
chy (melt curve peak, biu 4 [2]), trong biu ny trc honh (X) vn l bin
thin nhit ca bung , nhng trc tung (Y) th thay bin thin cng hunh
quang (RFU) thnh bin thin t l gim cng hunh quang so vi tng nhit
ca bung nhit (DRFU/DT). Nh s thay i ny, chng ta s thy tr s ca
DRFU/DT hu nh khng bin i khi s gia tng nhit ca bung nhit cha t
n Tm, nhng khi nhit bung nhit t n Tm th s c s gia tng ng k tr
s DRFU/DT, v trn biu chng ta s thy mt nh (peak) ca tr s ny. Gi tr
48
nhit bung tng ng vi nh ca ng biu din ny cho ta c tr s Tm
ca sn phm khuch i c trong ng phn ng. Nh vy vi phn tch melt curve
trong phng php real-time PCR s dng SYBR Green I, ngi lm th nghim c th
phn bit c sn phm khuch i c trong ng phn ng l t DNA ch hay l t
dimer primer, do m khc phc c nhc im khng c hiu ca cht pht
hunh quang ny trong pht hin sn phm khuch i xut hin trong ng phn ng
trong qu trnh lun nhit.

K thut phn tch phn gii cao nhit chy (High resolution melting, HRM)

Mc d c u im hn ethidium bromide, nhng SYBR green I cng cn mt

nhc im rt quan trng, l tn hiu hunh quang pht ra khng cao v li c th
c ch PCR. Chnh v nhc im ny m ng biu din nh chy s khng cao,
phn gii cc nh chy ca cc sn phm khuch i c trnh t khc bit hay/v chiu
di khc bit cng rt thp, khng th phn bit c chng vi nhau. Chnh v vy
m real-time PCR s dng SYBR I lm cht pht hunh quang khng th ng dng
trong multiplex real-time PCR, trong real-time PCR pht hin cc SNP (single
nucleotide polymorphism), hay real-time PCR nh c genotype.

Hin nay c nhng tin b ng k trong pht hin cc mu hunh quang chn
khc phc c cc nhc im ca SYBR green I. Cc mu ny c kh nng pht
hunh quang rt cao v hn na li hon ton khng c ch PCR nn phn gii ca
cc biu nh chy rt cao, do vy c gi l cc mu HRM (high resolution
melting dye). Bng 5 lit k mt s cht hunh quang HRM hin nay ang c cc
nh nghin cu ng dng, v s la chn mu no l ty thuc vo knh nh sng kch
thch v nh sng hunh quang m my real-time PCR h ang s dng c th thc
hin c trong qu trnh chy lun nhit. C th tm tt cc la chn ny nh sau:
SYTO9 hay LCGreen th ch dnh cho my real-time PCR Rotor Gene ca Corbett hay
Light Cycler ca Roche v cc my ny mi c knh mu thch hp; EVAGreen hay
BEBO th c th thch hp trn cc my real-time PCR thng dng v rt ti u trn
real-time PCR ca Biorad hay Applied Biosystem (dng ng knh mu FAM hay
SYBR green I); BOXTO th dng knh mu Joe c trn nhiu my real-time PCR
thng dng, c th kt hp vi real-time PCR dng Beacon probe gn cht pht hunh

49
 quang l FAM, nh vy m c th nh lng DNA ch bng FAM v phn tch HRM
curve vi knh Joe.
Bng 5: Trnh by cc cht hunh quang HRM hin nay ang c cc nh nghin cu s dng cng vi cc nng
thch hp phi pha trong PCR mix
Tn HRM Hng c bn quyn Bc sng kch thch Bc sng hunh quang Nng trong ng phn ng
pht ra

SYTO9 green Molecular Probes 485 498 2mM

LCGreen Idaho Technology 440-470 470-520 1-10mM
EVAGreen Biotium Corporate 470-495* 525-530* 1.33mM # 1X pha t 20X
BEBO Tataa Biocenter 468* 492* 1-5mM (3mM) # pha t 5mM

BOXTO Tataa Biocenter 515 552 0.3-0.7mM (0.5mM)

Ch thch hp cho Rotor Gene v Light Cycler; *Thch hp cho knh mu FAM v SYBR ca cc my real-time PCR thng dng;

Thch hp cho knh Joe ca cc my PCR thng dng

Vi real-time PCR dng HRM lm

mu chn, nh nghin cu cng nh ngi
lm th nghim hon ton c th thc hin
real-time PCR khng ch nh lng mt
cch c hiu c tc nhn ch c trong
mu th, m cn c th thc hin c
multiplex real-time PCR pht hin nhiu
tc nhn ch, pht hin cc t bin - thm
ch SNP vi ch 1 nucleotide khc bit, pht
hin v xc nh c genotype. Cha kha
chnh ca cc bc tin ny l kh nng
Biu 5:
phn bit c s khc bit khng ch v Biu HRM phn tch melt curve ca sn phm
khuch i ca real-time PCR dng EVAGreen
chiu di m c s khc bit trnh t c khi lm mu chn thc hin trn my real-time PCR
CFX96 ca Biorad, chng trnh HRM l c sn
n 1 nucleotide ca cc sn phm khuch trong my (Precision melt analysis sofware). Kt
qu cho thy kh nng phn bit rt r rng gia
khng t bin (ng biu din ), t bin T
i c trong ng phn ng nh bn cht u sang C (mu xanh bin) v heterozygote (mu
xanh l), c bit l khi biu hin th dng
vit ca cc mu HRM l khng c ch phn tch tc gim cng hunh quang
(difference RFU).
PCR v ng thi cng pht pht
hunh quang ca mt phn t mu HRM khi chn vo si i DNA c th tng n
trn 250 ln so vi khi t do trong dung dch. Chnh nh vy m ngi lm th nghim
khi chy chng trnh lun nhit phn tch melt curve phn gii cao (gi l chng
trnh HRM) tc l ch chy lun nhit phn tch melt curve trong mt khong cch
bit nhit hp (v d 73oC n 78oC l khong nhit m sn phm khuch t t

50
Tm, thay v 55oC n 95oC nh trong chng trnh lun nhit phn tch melve curve
bnh thng d Tm) kt qu biu nh chy hon ton phn gii cao phn
bit s khc bit ca cc sn phm khuch i c trong ng phn ng. Biu 5 minh
ha mt kt qu cho thy c th phn bit c s khc bit ch 1 nucleotide, k c tnh
trng heterozygote ca cc sn phm khuch i qua biu HRM.

2. Real-time PCR s dng probe lm cht pht hunh quang

Probe c dch l on d hay d, l nhng on oligonucletides si n

c trnh t c th bt cp b sung vi mt trnh t c hiu trn DNA ch (trong PCR,
l sn phm khuch i c hiu t DNA ch). S dng probe lm cht pht hunh
quang da trn nguyn tc l khi c mt sn phm khuch i c hiu trong ng phn
ng th s c s bt cp ca probe ln trnh t c hiu ca sn phm khuch i, v
khi c s bt cp ny th s c s pht hunh quang t ng phn ng khi n nhn c
ngun sng kch thch. C nhiu loi probe, v thm ch c primers c s dng lm
cht pht hunh quang cho real-time PCR. Trong phm vi bi ny, chng ti xin trnh
by k mt s thng c s dng hin nay.

a. Real-time PCR s dng Taqman probe

Nguyn tc k thut real-time PCR s dng Taqman probe

Trong k thut ny, real-time PCR mix ngoi cc thnh phn c bn ca PCR mix,
cn cha hai thnh phn quan trng probe c th pht hunh quang c khi c s
hin din ca sn phm khuch i c hiu t DNA ch, l: (1) Taqman probe, l
nhng oligonucleotides c trnh t b sung vi mt trnh t c hiu trn DNA ch, v
trnh t ny di khong 24 n 30 bases vi u 5 c gn cht pht hunh quang (gi
l reporter) cn u 3 c gn cht hp ph tng ng (gi l quencher) hp ph
c nh sng hunh quang c pht ra t reporter. (2) Enzyme Taq polymerase c
hot tnh 5-3 exonuclease c kh nng thy gii ct b probe khi probe ny bt cp
ln si khun v cn u 3 ca mi khi enzyme ko di mi tng hp si b sung. C
ch pht hunh quang ca Taqman probe trong cc chu k nhit c trnh by minh
ha trong hnh 25. C th tm tt nh sau: (1) Khi cha c s xut hin ca sn phm
khuch i c hiu t DNA ch th Taqman probe vn cn nguyn vn do vy m
hunh quang pht ra c t reporter u 5 s b quencher u 3 ca probe hp

51
ph, ng th nghim s khng pht c hunh quang khi nhn c ngun sng kch
thch. (2) Khi bt u c s xut hin sn phm khuch i c hiu th Taqman probe
s bt cp vo si khun ca sn phm ny giai on nhit bt cp v s b
enzyme Taq polymerase ct b ko di mi tng hp nn si b sung, do vy m
reporter ca Taqman probe s b ct ri xa quencher v pht c hunh quang khi
nhn c ngun sng kch thch. Cng nhiu sn phm khuch i xut hin th s
lng reporter t do s cng nhiu, n mt lc no cng hunh quang pht ra
t reporter mnh th my s ghi nhn c tn hiu hunh quang pht ra t ng phn
ng khi nhn c ngun sng kch thch.

Reporter v quencher ca Taqman probe

Nh ni trn, reporter l mt cht gn vo u 5 ca Taqman probe, c kh

nng pht c hunh quang khi nhn c ngun sng kch thch. C nhiu loi ha
cht c s dng lm reporter. Trnh by trong bng 6 l cc ha cht ny vi cc chi
tit cn thit nh di sng ca ngun sng kch thch, di sng ca hunh quang
pht ra, v cc quencher tng ng.

Quencher l cht c
nh sng kch thch

kh nng hp ph c [1] Khi cha c mt sn phm khuch i c hiu,

Taqman probe khng pht c hunh quang
khi nhn c ngun sng kch thch v hunh
quang pht ra b quencher hp ph ht
nh sng hunh quang R Q

pht ra t reporter khi

reporter nhn c nh
[2] (a) Khi c mt sn phm khuch i c hiu,
R Q
Taqman probe s bt cp trn trnh t c hiu
sng kch thch. C hai ca sn phm khuch i giai on nhit
bt cp sau giai on bin tnh

loi quencher, l
quencher pht hunh R Q
[2] (b) Taqman probe s tr thnh trnh t cn u
quang v quencher pht 3 khi Taq polymerase ko di mi bt u
tng hp si b sung

nhit. Quencher pht

R

hunh quang l quencher Q

[2] (c) Nh c hot tnh 5-3 exonuclease nn Taq
polymerase s ct b Taqman probe lm
khi hp ph nh sng reporter b ct ri xa quencher v nh vy m
hunh quang pht ra t reporter s khng cn b
quencer hp ph na
hunh quang t reporter s R
Q

chuyn nng lng hp [2] (d) Giai on pht hunh quang ca reporter xy
ra trong sut qu trnh Taq polymerase ko di
ph c thnh nh sng, mi tng hp si b sung

do vy quencher s pht ra Hnh 25: Tm tt c ch hot ng ca Taqman probe trong real-time PCR

52
hunh quang nhng khng n c CCD hay cm bin quang v b cn li bi knh
lc ch dnh cho nh sng hunh quang pht ra t reporter. TAMRA l mt v d minh
ha cho quencher pht hunh quang, thng c dng cho taqman probe c reporter
l FAM (bng 6). Dng quencher pht hunh quang s c mt tr ngi l hunh quang
pht ra t quencher c th trng vi ph hunh quang ca reporter ca mt Taqman
probe khc, nu real-time PCR c thit k multiplex, do vy m khi chn lc
reporter cho probe th 2 hay th 3 th nh nghin cu phi trnh tr ngi ny.
Quencher pht nhit l quencher s chuyn nng lng hp ph c thnh nhit, v
d DABCYL hp ph hunh quang bc sng 425, BHQ0 hp th hunh quang bc
sng 430-520 (ti u 493), BHQ1 hp th hunh quang 480-580 (ti u 534), BHQ2
hp th hunh quang 560-670 (ti u 579), BHQ3 hp th hunh quang 620-730 (ti
u 672). Quencher pht nhit hin nay c a chung hn quencher pht hunh
quang v kh nng hp ph hunh quang rt mnh, cc BHQ c xem nh cc l en
(black hole quencer, BHQ) ht ly tt c cc hunh quang t reporter khng cho bt c
hunh quang no pht ra c t reporter, ng thi rt d dng thit k cc taqman
probe dng cho multiplex PCR m khng lo n ph hunh quang ca cc reporter c
b trng vi ph hunh quang ca quencher v quencher khng h pht hunh quang.
Bng 6: Trnh by cc reporter hin nay ang c cc nh nghin cu s dng cng vi bc sng kch thch,
hunh quang pht ra, v quencher tng ng
Tn reporter Bc sng Bc sng Quencher Tn reporter Bc sng Bc sng HQ Quencher
kch thch HQ pht ra kch thch pht ra

FAM 495 520 TAMRA, DABCYL, BHQ1 Cy3TM 548 566 BHQ2

TAMRA 557 583 BHQ2, DABCYL Cal FluorGold 540 522 544 BHQ1

TET 521 536 BHQ1, DABCYL Cal Fluor Orange 560 538 559 BHQ1

TEXAS RED 590 610 BHQ2, BHQ3, DABCYL Cal Fluor Red 590 569 591 BHQ2

VIC 538 554 BHQ1 Cal Fluor Red 610 590 610 BHQ2

HEX 535 556 BHQ1, BHQ3, DABCYL Cal Fluor Red 635 618 637 BHQ2

JOE 529 555 BHQ1 LC red 640 618 637 BHQ2

Cy5 647 667 BHQ2, BHQ3 Pulsar 650 460 650 BHQ2

Cy5.5 690 705 BHQ2 Quasar 670 647 667 BHQ2

Rox 586 610 BHQ2, BHQ3, DABCYL Quasar 705 690 705 BHQ3

Thit k mi v Taqman probe

Thit k mi cho real-time PCR dng Taqman probe cng theo cc qui lut thit
k mi chung cho PCR (trnh by trong phn thit k mi dnh real-time PCR dng
mu hunh quang chn). Trong real-time PCR dng Taqman probe th nn thit k mi
c nhit chy khong 55oC - 60oC v thp hn nhit chy ca Taqman probe
53
khong 5oC - 10oC. thit k Taqman probe, nn thit k khng di qu 30 bases,
t l GC trong probe khong 30-80 % vi C nhiu hn G, v tr bt cp ca u 5 ca
Taqman probe ch 2-5 bases cch v tr 3 ca mi bt cp trn cng si ch, v rt
quan trng l u 5 gn reporter khng phi l G v G c th l quencher cho rt nhiu
reporter. C th s dng phn mm Beacon Designer thit k mi v Taqman probe.
Phn mm ny c cp min ph cho cc phng th nghim mua my PCR iCycler
cua Biorad. Ngoi ra, vic chn reporter v quencher cng l mt vn ht sc quan
trng. Xin tham kho bng 6 c nhiu kh nng la chn cho ph hp.

Khi thit k mi cho real-time PCR dng Taqman probe th cng nn lu

chiu di sn phm khch i trong khong 75-150 bps, khng nn qu 150 bps hiu
qu PCR c th t c ti ho. Tuy nhin trn thc t cng cn ty thuc vo DNA
ch c cho php chng ta chn c mi t c ti ho c sn phm khuch i
t nh vy hay khng. Cng ty Nam Khoa c b th nghim RT real-time PCR s
dng Taqman probe pht hin v nh lng HCV-RNA da trn s khuch i si
cDNA ch di 240 bps phin m ngc t vng 5NC ca b gene ca virus m vn
t c hiu qu PCR ti ho th hin qua E% lun t 90-105 % (biu 6); cng
chnh nh vy m cng ty trin khai c th nghim gii trnh t sn phm real-time
PCR ny xc nh c genotype ca HCV nhim trn bnh nhn.

Chng trnh lun nhit

real-time PCR dng taqman
probe thng ch c hai giai
on nhit l: bin tnh
94 95oC trong 15 30
giy, k l giai on va
bt cp va ko di 60oC
trong 30-60 giy v thit b
real-time s hot ng giai Biu 6: Biu chun ca RT real-time PCR s dng Taqman
probe pht hin v nh lng HCV-RNA da trn mi
on ny. giai on nhit khuch i trnh t 240bps trn vng 5NC ca b gene
HCV
60oC th Taqman probe
s bt cp vo si ch trc v y l nhit bt cp ti ho ca Taqman probe (thp
hn Tm ca Taqman probe l 65 70oC, nhng bng hay cao hn Tm ca mi l 55oC

54
- 60oC) ri sau mi mi bt cp vo. Chnh nh vy m khi Taq polymerase tng
hp si b sung, enzyme ny mi c c hi thy gii c Taqman probe cn u n.
Nu mi bt cp trc vo si ch th s khng c c hi Taqman probe bt cp
vo si ch v nh vy th Taqman probe s khng th b thy gii khi enzyme Taq
polymerase tng hp si b sung. y chnh l l do gii thch ti sao phi thit k
mi c Tm khong 55-60oC v thp hn Tm ca Taqman probe 5oC - 10oC.

Pha PCR mix cho real-time PCR dng Taqman probe

Pha PCR mix cho real-time PCR dng Taqman probe bao gm cc thnh phn vi
cc nng hay hm lng nh sau: (1) PCR buffer 1X, (2) Taq polymerase c hot
tnh 5-3 exonuclease 1.25 hm lng 2 unit cho mt th tch phn ng, (3) dNTP 200
mM/each, (4) mi xui v mi ngc hm lng 10-25 pm cho mi th tch phn ng,
(5) MgCl2 5 mM, (6) Taqman probe 2-5 pm cho mi th tch phn ng; (7) Ngoi ra c
th thm dUTP, UNG vi nng hay hm lng nh cc pha cc PCR mix real-time
hay PCR mix khng real-time khc, v (8) mu hunh quang nn nu thit b real-time
yu cu. Nn thm d hm lng ca Taqman probe c cng hunh quang
c ghi nhn l cao nht, khng phi cho hm lng ca Taqman probe cao th s lm
cng hunh quang pht ra s cao m c khi c tc ng ngc li v s lm cc
phn t Taqman probe qu gn nhau nn hunh quang pht ra t reporter ca probe b
thy gii vn b cc quencher ca cc Taqman probe khc hp ph.

Chn reporter ca Taqman probe cho multiplex PCR

thit k c cc Taqman probe dng cho multiplex PCR vi cc quencher l

BHQ th yu t quan trng nht l phi la chn nh th no bc sng kch thch
cc reporter, v nht l bc sng hunh quang pht ra t cc reporter phi cch xa
nhau tn hiu hunh quang c thit b ghi nhn cho tng reporter s khng b nh
hng ln nhau. Ngoi ra trn thc t s la chn reporter l cn ty thuc vo cc
knh lc mu c trn thit b real-time na. Biu 7 minh ha mt la chn c
xem l ti u cho multiplex 5 mu thc hin vi real-time PCR trn iQ5 ca Biorad.
Trong s la chn ny, chng ta thy bc sng hunh quang ti a ca tt c 5
reporter l cch xa nhau nht v tt c u ph hp vi 5 knh knh lc ngun sng
kch thch n v hunh quang pht ra t reporter (trnh by trong bng 7).

55
Bng 7: Lit k cc knh knh lc ngun sng kch thch v knh lc hynh quang c trn iQ5 tng ng vi cc
reporter khuyn co s dng. Cc ch in m l reporter ti u c chn cho 5 mu

Tn reporter Bc sng Knh knh lc Bc sng Knh knh lc

kch thch ngun sng kch thch HQ pht ra hynh quang

FAM 495 Channel 1: 475495 nm 520 Channel 1: 515545 nm

TAMRA 557 Channel 3: 530560 nm 583 Channel 3: 575595 nm
Cy3 548 Channel 3: 530560 nm 566 Channel 2: 565585 nm
TEXAS RED 590 Channel 4: 560590 nm 610 Channel 4: 610640 nm
Rox 586 Channel 4: 560590 nm 610 Channel 4: 610640 nm
TET 521 Channel 2: 515545 nm 536 Channel 1: 515545 nm
VIC 538 Channel 2: 515545 nm 554 Channel 2: 565585 nm
HEX 535 Channel 2: 515545 nm 556 Channel 2: 565585 nm
JOE 529 Channel 2: 515545 nm 555 Channel 2: 565585 nm
LC640 618 Channel 5: 615645 nm 637 Channel 4: 610640 nm
Cy5 647 Channel 5: 615645 nm 667 Channel 5: 670700 nm
TAMRA

TAMRA
TEXAS

TEXAS
HEX

HEX
FAM

FAM
RED

RED
Cy5

Cy5
Cng HQ

Cng HQ

Bc sng kch thch Bc sng hunh quang

Bc sng l = nm Bc sng l = nm

Biu 7: Minh ha bc sng kch thch v bc sng hunh quang ca mt chn la multiplex 5 mu trn
iQ5. Chn la ny c xem l thch hp nh cch xa nhau gia cc bc sng kch thch ti
a v bc sng hunh quang ti a ca cc reporter

b. Real-time PCR s dng Beacon probe

Trong k thut ny, probe c s dng c tn l Beacon molecular probe. y l

probe c trnh t di khong 25-40 bases, c u 5 gn vi reporter v u 3 gn vi
quencher. Probe c trnh t 15-39 bases gia l b sung vi mt trnh t c hiu trn
mt si ca DNA ch, cn hai u ca probe th mi u c mt trnh t 5-6 bases b
sung vi nhau lm cho probe to thnh cu trc hnh kp tc do trnh t ca hai u bt
cp nhau. C ch hot ng ca Beacon probe c minh ha trong hnh 26, tm tt
nh sau: (1) giai on nhit bin tnh, cu trc kp tc ca Beacon probe khng
cn nn nu giai on ny reporter nhn ngun sng kch thch th n s pht hunh
quang. (2) giai on nhit bt cp, nu cha c mt sn phm khuch i c
hiu, Beacon probe s khng pht c hunh quang khi nhn c ngun sng kch

56
thch v cu trc kp tc ca hai u
R Q

lm cho reporter gn vi quencher khin

tt c hunh quang pht t reporter u b
quencher hp ph. Nhng nu c mt sn
phm khuch i c hiu, Beacon probe
Khi khng c mt sn phm khuch i c hiu, giai an
nhit bt cp, Beacon probe s khng pht c hunh
quang khi nhn c ngun sng kch thch v cu trc chn tc
ca hai u lm cho repoter gn vi quencher khin tt c
hunh quang pht t reporter u b quencher hp ph.

s bt cp vo trnh t c hiu trn si

ch ca sn phm khuch i, nh vy
reporter cch xa quencher nn reporter
pht c hunh quang khi nhn ngun
sng kch thch. (3) Trong giai on nhit
ko di, enzyme Taq polymerase
R Q
Khi c mt sn phm khuch i c hiu, giai on nhit
bt cp, Beacon probe bt cp vo trnh t c hiu trn si ch
ca sn phm khuch i, nh vy reporter cch xa quencher
nn reporter pht c hunh quang khi nhn ngun sng kch
thch.
R
Q

khng thy gii Beacon probe m ch lm

tch Beacon probe khi si ch khi si
b sung c tng hp ko di n trnh Trong giai an nhit ko di, enzyme Taq polymerase c
s dng khng thy gii Beacon probe m ch lm tch Beacon

t m probe bt cp vo. Cui giai on probe khi si ch khi si b sung c tng hp ko di n

trnh t m probe bt cp vo.

ko di, Beacon probe tch khi si ch R Q

v tr li cu trc kp tc nn khng cho

reporter pht c hunh quang nu nhn
c ngun sng kch thch.

Beacon probe c mt s u im,

Cui giai an ko di, Beacon probe tch khi si ch v tr li
cu trch chn tc nn khng cho reporter khng c hynh
l: (1) rt c hiu v ch cn trnh t si quang nu nhn c ngun sng kch thch

khun khc bit l s khng c bt cp v Hnh 26: Tm tt c ch hat ng ca Beacon

probe trong real-time PCR
nh vy l s khng c hunh quang -
chnh nh vy Beacon rt u vit pht hin SNP; (2) rt tt thc hin multiplex
pht hin cng lc nhiu tc nhn ch. Tuy nhin, Beacon tng i kh thit k, nht
l khi thit k trnh t hai u 5 v 3 phi ch c s bt cp mnh lm cho
probe c cu trc kp tc m khng lm cho cu trc ca probe t gp li thnh cu
hnh khng kp tc, v nh vy s lm cho hunh quang ca reporter pht ra t do
khng mong mun. Ngoi ra s bt cp ca trnh t hai u cng khng c qu
mnh khng ngn cn probe bt cp vo si ch khi c mt ca si ch. Chng
trnh Beacon designer c cung cp min ph khi mua iCycler ca Biorad l mt

57
trong cc chng trnh rt tt, gip ngi s dng d dng thit k Beacon probe trn
trnh t si khun nhp vo.

c. Real-time PCR s dng probe lai (hybridization probes)

Trong k thut ny, ngi ta dng 2 probe. Probe D A

lai 1 c u 3 gn mt cht pht hunh quang D
(donnor), v probe lai 2 c u 5 gn mt cht pht
hunh quang A (acceptor). trnh khng cho probe
Khi cha c mt sn phm khuch i c hiu, hunh
quang pht ra t D ca probe lai 1 khng kch thch
c A ca probe lai 2 v chng cch xa nhau
D A

lai 2 ny c th ko di khi n bt cp ln si khun,

ngi ta thng gn thm mt gc phosphate u 3. Khi c mt sn phm khuch i c hiu, Probe lai 1
v probe lai 2 bt cp ln si khun lm cho D rt gn
A nn hunh quang pht ra t D (c bc sng trng

Hai probe lai ny c thit k sao cho khi bt cp trn vi bc sng ngun sng kch thch ca A) s c
A hp ph ri chuyn sang pht hunh quang vi bc
sng khc bit vi bc sng pht hunh quang ca D

si khun th u 3 ca probe lai 1 ch cch u 5 ca

probe lai 2 khong t 3-5 bases, ngoi ra phi chn D D

v A sao cho hunh quang t D pht ra c di sng A

trng vi ngun sng kch thch ca A lm cho A

pht ra c hunh quang c di sng khc bit vi
hunh quang pht ra t D. h thng hot ng c D A

th thit b realtime phi pht c ngun sng kch

thch D, nhng CCD hay cm quang phi c knh lc
Trong giai on ko di, Taq polymerase v khng c
ch ghi nhn hunh quang pht ra t A ch khng hot tnh 5-3 exonuclease nn s tch cc probe lai 1
ri probe lai 2 khi si khun, do vy l A tch ri D
nn D khng cn nhn c bun sng kch thch t D
na v A s khng cn pht hunh quang c.
phi t D. C ch hot ng ca hybridization probes
Hnh 27:
c tm tt nh sau (hnh 27): giai on nhit Tm tt c ch hot ng ca probe
lai trong real-time PCR
bt cp, nu khng c sn phm khuch i c hiu th
D ca probe lai 1 pht hunh quang do nhn c ngun sng kch thch, nhng thit
b real-time khng ghi nhn c hunh quang ny v bc sng pht ra t D khng i
qua c knh lc n CCD camera hay cm quang. Nu c sn phm khuch i
hin din th probe lai 1 v probe lai 2 bt cp c vo si khun, do vy m hunh
quang pht ra t D s l ngun sng kch thch A, lm cho A pht hunh quang v thit
b real-time s ghi nhn c hunh quang ny nu lng sn phm khuch i t
ngng hunh quang pht ra t A qua c knh lc n c CCD camera hay
cm quang ca thit b real-time. C ch hot ng ca probe lai nh vy nn k thut
ny cn gi l k thut FRET real-time (Fluorescence Resonance Energy Transfer).

58
 Cng nh tt c cc c ch pht hunh quang khc ca real-time PCR, k thut
real-time PCR s dng cc probes lai c ng dng nh lng tc nhn ch c
trong mu. Ngoi p dng ny, real-time PCR s dng cc probe lai cn c mt u
im, l ng dng nh genotype ca tc nhn ch hay l nh c s khc bit
ch mt nucleotide (SNP) ca cc tc nhn ch. Nguyn tc ca ng dng ny l nhit
chy ca hai probe lai trn cc si khun khc bit v trnh t d ch mt nucleotide
cng s khc nhau, nn lm cho ng biu din nh chy ca chng cng khc nhau.
Hnh 28 v biu 8 l mt minh ha hiu r hn ti sao real-time PCR vi probes
lai c th gip phn bit c cc khc bit ca tc nhn ch v genotype hay thm
ch ch mt SNP. c th thc hin c ng dng ny th ngi lm th nghim s
chy thm mt chng trnh phn tch melt-curve sau khi my hon tt chng trnh
lun nhit real-time.

nh chy nh chy
ca A ca B
DRFU/DT C
o

D A o D A
78 C

A
D A o D
79 C

A
D A D
o
80 C

D A D A

o
81 C o
T C
o o o o o o
Sn phm khuch i B Sn phm khuch i A 78 C 79 C 80 C 81 C 82 C 83 C

Hnh 28: Biu 8:

Minh ha cho thy sn phm khuch i A ch khc B Minh ha cho thy nh nhit chy ca A phn bit
mt nucleotide nhng nhit chy ca probe lai trn r vi nh nhit chy ca B. Nh vy c th thy
si khun khc bit c A v B khc bit nhau

d. Cc k thut real-time PCR s dng probe khc

Phn trn l gii thiu tng i chi tit v 3 phng php real-time PCR s dng
probes lm cht pht hunh quang. y l nhng phng php thng c s dng
trong nhiu nghin cu hay l trong chn on lm sng. Ngoi 3 phng php dng
probe lm cht pht hunh quang ny, cn c nhiu phng php dng probes hay c
primer na lm cht pht hunh quang, nh Eclipse probe, Amplifluor primers,
Scorpion primers, Lux primers, BD-QZyme primers...Xin tham kho ti liu Real-time

59
 PCR Applications Guide (http://www.gene-quantification.de/real-time-pcr-guide-bio-
rad.pdf) r hn.

Cc ng dng ca real-time PCR

1. nh lng tc nhn ch c trong mu th

c trng ca real-time PCR l pht hin sn phm khuch i trong qu trnh

chy PCR khi sn phm khuch i t DNA ch c nhn bn t s lng
lm cho ng phn ng pht c hunh quang khi nhn c ngun sng kch thch.
Chnh nh c trng ny m ngi lm th nghim c th bit c s lng bn DNA
ch ban u c trong ng phn ng da vo s xut hin hunh quang ca ng phn
ng sm hay mun, tc l chu k ngng (Ct) ca ng phn ng nh hay ln. C hai
cch nh lng tc nhn ch da vo mc ch nh lng m ngi lm th nghim
quan tm:

a. nh lng tuyt i

Ngi lm th nghim s s dng nh lng tuyt i nu mc ch ca xt

nghim l nh lng bit r s copies ca tc nhn ch c trong mu th hay trong
bnh phm. V d xt nghim nh lng virus vim gan B, hay vim gan C, hay HIV
trong mu mu ca bnh nhn theo di hiu qu iu tr c hiu. Phng php nh
lng ny i hi phi bit r lng (th tch, hay c th l trong lng) ca mu th.
c th thc hin phng php nh lng tuyt i, nh nghin cu hay ngi lm
th nghim phi thc hin xt nghim real-time PCR ca mu th cng lc vi cc mu
chun bit trc s lng ri tnh ra s copies DNA ca tc nhn ch c trong ng

Biu 9: Biu chun l mt yu t quan trng lm c nh lng tuyt i. Nh biu chun m

chng trnh ca my c th tnh c hm lng DNA ch ban u c trong ng phn ng.

60
phn ng da vo ng bin din chun (minh ha biu 9) xc nh mi quan h
gia chu k ngng (Ct) vi s lng copies DNA ch ban u c trong ng phn ng.
Cui cng xc nh s copies tc nhn ch c trong mu th hay bnh phm da vo
h s pha long mu v h s tch chit DNA hay RNA ca phng php chun b
mu trc khi thc hin PCR.

C th phng php tnh ton s copies ca DNA ch ban u c trong ng phn

ng da vo ng biu din chun nh sau:

ng biu din chun cho c mt hm s biu th mi tng quan gia

chu k ngng (Y = Ct) vi log10 ca s lng bn DNA ch ban u c trong
ng phn ng (X = log10 Sq). Hm s l: Y = [slope (X)] + intercept, v cc
thng s slope v intercept u hin th trn biu chun.

T hm s ny, ngi lm th nghim s hiu c ti sao my tnh hin th

c Sq, l nh tnh ton t Sq = 10[(Ct intercept)/slope].

b. nh lng tng i

Nu mun nh lng tc nhn ch m ngi lm th nghim khng th cn o

ong m c mu th c c s lng chnh xc th nn lm phng php nh
lng tng i. V d xc nh tm s b nhim mt loi virus gy bnh no nhiu
hay t; trong trng hp ny ngi lm th nghim u c th cn chnh xc s lng
tm post c nghin th nghim do vy m nu dng phng php nh lng
tuyt i s gp tr ngi v nu lng tm th nghim nhiu qu th lng nhim virus
d t vn c th cho con s nh lng tuyt i cao v ngc li nu lng tm th
nghim qu t th con s nh lng tuyt i c th thp d tm nhim virus cao. nh
lng tng i cng c s dng trong cc nghin cu v ung th nh lng
mt bin hin gene no , hay cng c s dng trong nh lng sn phm bin i
gene. Cc v d minh ha trong cc phng php nh lng tng i sau y s lm
r hn v nh lng tng i.

nh lng tng i da trn n vi khi lng (unit mass)

Phng php nh lng ny c th p dng trong nh lng biu hin gene vi

lng mu th nghim phi c xc nh chnh xc (lng t bo, lng nucleic acid
tch chit t mu...). Ly v d nh lng biu hin gene P53 trn cy t bo c th

61
cht nghin cu X gy ung th so vi cy t bo chng khng th cht nghin cu X.
Nh nghin cu thc hin hai l cy t bo cng ngy tui, sau mt l th thm cht
X, mt l chng khng th cht X. Sau ly cng mt lng t bo ca c hai l
tch chit v nh lng RNA ton b ca c hai l. Ly cng mt lng RNA tch
chit c ca c hai l (v d 50 ng cho mi l), thc hin th nghim RT real-time
PCR vi mi c hiu mRNA ca P53. Xc nh Ct ca mRNA ca P53 vi gi tr
Ct[T] l chu k ngng ca khuch i mRNA ca P53 trn 50 ng RNA tch chit t
l t bo c th cht X v Ct[C] l chu k ngng ca khuch i mRNA ca P53 trn
50 ng RNA tch chit t l t bo chng khng c th cht X. Nu hiu qu PCR l t
100 %, ngha l c sau mi chu k nhit lng DNA lun tng gp i (E = 2) th t l
biu hin gene P53 ca l t bo c th cht X so vi l chng s l:

T l biu hin gene P53 [Th/Chng] = 2(Ct[C]-Ct[T])

V d, nu Ct[T] l 12, Ct[C] l 15, th t l biu hin gene P53 ca t bo c th

cht X so vi t bo khng th cht X l 2(15-12) = 23 = 8. C ngha l cht X lm cho
biu hin P53 trn t bo tng gp 8 ln so vi chng.

nh lng tng i da trn gene tham chiu (reference gene)

Phng php nh lng ny c th p dng trong nh lng tc nhn gy bnh

trong cc mu th m khng th xc nh chnh xc s lng, hay l nh lng biu
hin gene trong cc m ung th. C th p dng mt trong cc phng php sau y:

Phng php 2-DDCt ca Livak

Phng php ny c th dng nghin cu biu hin mt gene trong mu cy t
bo ung th, so vi mu cy t bo bnh thng; hay cng c th nghin cu biu hin
mt gene no trn cy t bo c th cht gy ung th X so vi cy t bo khng th
X nh v d trn. Trong phng php ny, ngi lm th nghim s nh lng khng
ch gene cn nghin cu (Target = Tg) m c mt gene tham chiu (reference = Ref)
tc l gene m cc nghin cu trc xc nh l c biu hin nh nhau gia hai
mu t bo (Th: t bo ung th hay c th cht X, gi l mu T; v Chng: t bo
khng ung th hay khng th cht X, gi l mu C). Nh vy ng vi mu T v mu C
s c cc kt qu nh lng cho c gene Tg v gene Ref qua cc thng s:

62
 Ct(T/Tg): l chu k ngng ca gene ch (Tg) trong mu th (T),
Ct(T/Ref): l chu k ngng ca gene tham chiu (Ref) trong mu th (T),
Ct(C/Tg): l chu k ngng ca gene ch (Tg) trong mu chng (C), v
Ct(C/Ref): l chu k ngng ca gene tham chiu (Ref) trong mu chng (C).
Da trn cc thng s ny, chng ta s thng ha (normalized) chu k ngng
ca gene ch trn mu th (T) v mu chng (C) bng cch tnh hiu s chnh lch Ct
ca gene ch vi gene tham chiu trn cc mu:
Mu th: DCt(T) = Ct(T/Tg) - Ct(T/Ref)
Mu chng: DCt(C) = Ct(C/Tg) - Ct(C/Ref)
Sau s thng ha DCt mu th bng hiu s chnh lch DCt(T) vi DCt(C)
DDCt = DCt(T) - DCt(C)
T tnh ra t l biu hin ca gene ch trn mu th so vi mu chng, nu
hiu qu PCR ca c hai u t 100%, l R = 2-DDCt

V d sau y minh ha cho phng php 2DDCt ca Livak p dng trong nghin
cu biu hin gene P53 trn cy t bo bung trng ung th (T) so vi t bo bung
trng bnh thng (C). C hai u c tch chit RNA ton b ri ly 50 ng RNA
c tch chit lm th nghim RT real-time PCR vi 2 Taqman probe v mi c
hiu cho mRNA ca P53 v ca gene tham chiu l GAPDH (cc nghin cu trc
chng minh l GAPDH biu hin khng khc bit trn m bnh thng v m ung th).
Kt qu trnh by trong bng 8 di y:
Bng 8: Chu k ngng ca gene P53 (Tg) v GADPH (Ref) ca hai mu cy t bo bung trng ung th (T) v
bnh thng (C).

Mu cy t bo Ct P53 (Tg) Ct GAPDH (Ref)

Bnh thng (C) 15.0 16.5

Ung th (T) 12.0 15.9

Vi cc kt qu trnh by trong bng 8 ny, chng ta s tnh c

DCt(T) = Ct(T/Tg) - Ct(T/Ref) = 12.0 15.9
= -3.9
DCt(C) = Ct(C/Tg) - Ct(C/Ref) = 15.0 16.5
= -1.5
DDCt = DCt(T) - DCt(C) = -3.9 (-1.5) = -2.4

63
 T tnh ra t l biu hin ca P53 trn mu th so vi mu chng, nu hiu qu
PCR ca c hai u t 100%, l R = 2-(-2.4) = 5.3
C ngha l P53 t bo ung th biu hin gp 5.3 ln t bo bnh thng.

Phng php Pfaffl

Phng php 2DDCt ca Livak p dng trn real-time PCR m hiu qu PCR ca c
hai gene ch v tham chiu u t 100% tc l E = 2. Nu hiu qu PCR ca hai
gene khc nhau, gene ch (Tg) l E(Tg), v gene tham chiu (Ref) l E(Ref) th chng
ta phi s dng mt cch tnh tng qut hn ca phng php Pfaffl tnh t l (R)
biu hin ca gene ch trn mu th so vi mu chng:
R = E(Tg)DCt(C/Tg-T/Tg)/E(ref)DCt(C/Ref-T/Ref)
Cng thc ny tht ra ch l dng tng qut ca cng thc Livak, v nu c hai
gene u c hiu qu PCR 100%, tc E = 2, th cng thc trn s l:
R = 2DCt(C/Tg-T/Tg)/2DCt(C/Ref-T/Ref) = 2DCt(C/Tg-T/Tg) - DCt(C/Ref-T/Ref)
= 2-[DCt(T/Tg-C/Tg)] [DCt(T/Ref-C/Ref)] = 2-DDCt
Minh ha bng cch p dng vo v d trn, nu c hai gene u c hiu qu PCR
l 100% (E = 2) th cng thc trn s c tnh l:
R = 215-12/216.5-15.9
= 23/20.6 = 8/1.52 = 5.3

nh lng tng i da trn khi lng vt ch

c th nh lng c t l nhim tc nhn virus gy bnh trn tm nhiu hay

t th phi xc nh c lng mu c th nghim. Do vy cn phi c hai h thng,
mt l h thng th nh lng c tc nhn virus gy bnh c trong mu th, v
mt l h thng tham chiu nh lng c lng mu a vo th nghim. Vi
phng php real-time PCR th thng s nh lng s c c chnh l chu k
ngng (Ct), v t l nhim tc nhn virus gy bnh s chnh l t l % Ct ca ng
biu din khuch i ca mt an gene c hiu tc nhn ch (gi l Ct[T]) so vi Ct
ca ng biu din khuch i ca mt an gene gi nh hin din trong b gene vt
ch (gi l Ct[R]). Chng ti t tn t l ny l IP (Infected pathogen).

i vi h thng th th nguyn tc ca k thut real-time PCR l khuch i v

ng thi pht hin mt an DNA c hiu t b gene ca tc nhn virus gy bnh

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nh s dng mt cp mi c hiu v mt Taqman probe c hiu c gn cht pht
hynh quang FAM u 5 v cht hp ph hynh quang u 3. Trong khi chy
PCR, mt khi c sn phm khuch i c hiu xut hin trong ng PCR th taqman
probe c hiu vi trnh t ch s bt cp vo sn phm khuch i v s b thy gii
bi men taq polymerase (nh hot tnh 5- 3 exonuclease) khi tng hp si b sung
giai an ko di. S thy gii taqman probe s lm tch ri cht pht hynh quang
FAM u 5 khi cht hp ph hynh quang (quencher) u 3 ca probe, nh vy
ng phn ng s pht hynh quang khi nhn c nh snh kch thch, v s pht
hynh quang ny s c ghi nhn bi u c real-time ca my. Mu th cha nhiu
bn ch ban u s c chu k ngng (Ct = chu k m u c realtime ca my bt
u ghi nhn c c tn hiu hynh quang trong ng phn ng) pht hin sm hn l
mu th c t bn ch. i vi h thng ch th th cng tng t nh vy nhng mi
v Taqman probe th s c thit k c hiu cho mt gene gi nh hin din trong
b gene ca vt ch. y, trnh s cnh tranh ln nhau gia hai h thng th v
tham chiu lm cho s nh lng khng chnh xc, chng ti thit k hai h thng ny
trong hai ng phn ng khc nhau, do vy m Taqman probe ca h thng tham chiu
cng s s dng cng mu pht hynh quang FAM u 5 ging nh Taqman probe
ca h thng th.

V d nh lng virus gy mang (GAV, Gill associated virus) nhim trong

mu tm bng b th nghim RT qPCR pht hin v nh lng GAV do cng ty Nam
Khoa sn xut. Nguyn tc ca b th nghim ny, l nh lng GAV ng thi vi
nh lng gene tham chiu l cytochrome b ca tm c trong mu xc nh c t
l tm nhim bnh. Phng php thc hin l:

(1) Trc ht ly mt lng tm th nghim khng cn chnh xc s lng, thc

hin tch chit RNA tan phn ri a vo tng hp cDNA vi mi ngu nhin;

(2) Sau thc hin real-time PCR s dng Taqman probe v mi c hiu tng
ng cho GAV v cho gene cytochrome b. Kt qu s cho chng ta Ct[GAV] ca GAV
v Ct[Cytob] ca gene cytochrome b. T l Ct[GAV]/Ct[Cytob] cho bit t l nhim
GAV trn mu tm.

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2. Xc nh t l bin i gene (GMO, Gene Modified Organism) c trong mu

Li im chnh yu ca sinh vt bin i gene chnh l lm tng gi tr dinh dng

v/hay khng li su bnh hay thuc dit c. u, bp, la m, la v bng l cc cy
lng thc thng c bin i gene nht. Tuy c li im nhng mc an tan
vn cha c bit r nn nhiu quc gia i hi kim sot t l bin i gene ca sinh
vt v sn phm ca n, v chnh do vy m cn phi c cc phng php chnh xc v
nhy cm lm c vic ny. Cc phng php hin nay ang c s dng l
ELISA v PCR. ELISA tng i r v nhanh, nhng phng php PCR c li im l
nhy cm, v c th nh lng c. C s ca PCR l nh lng mt promoter
thng c trong vector biu hin c gene chn vo vector ny sau khi chuyn
gene vo sinh vt ch. V d u bin i gene khng c thuc tr c l do nhn
c gene gip phc hi mt ng bin dng acid amin thit yu vn d b
glyphosate ph hy. y chnh l gene enolpyruvylshikimate-3-phosphate synthase
(epsps) c c lp t vi khun c t, l Agrobacterium sp. dng cp4. Gene ny
c chn vo vector l virus khm sp-l (cauliflower mosaic virus, CaMV) c mang
promoter 35S ri chuyn vo cy u. Vector c mang promoter 35S ny rt thng
c dng chuyn gene trong thc vt l v ph chuyn gene v biu hin c
gene rt rng cho nhiu lai cy. Ngoi ra promoter 35S c trnh t rt n nh v c
bit r. Chnh v vy nn CaMV 35S promoter l i tng chnh yu dng nh
tnh v nh lng t l bin i gene trong cc sinh vt hay sn phm bin i gene.
Sau y l mt v d nh lng t l bin i gene trong u nnh minh ha.

thc hin c th nghim, trc ht phi c cc mu chun tc l mu u

nnh c trn thm u nnh bin i gene theo t l bit trc t 0% n 5% (mua
t Sigma). Gene ch nh lng l promoter 35S, gene tham chiu l gene lectin
ca u nnh. Cc mu chun v mu th u c tch chit DNA (dng b tch
chit DNA cho thc vt, nh b DNeasy plant mini kit ca Qiagen c th tch chit
c 1-2mg t 55-100mg sn phm). Thc hin real-time PCR (s dng SYBR hay
Taqman probe) nh lng hai gene trn mt lng nht nh DNA tch chit c t
cc mu th v mu chun. Kt qu Ct c ghi nhn v trnh by trong bng 9. Biu
10 trnh by mi quan h tuyn tnh gia t l GMO% v DCt.

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Bng 9: Chu k ngng ca S35 v lectin ca cc log(GMO%)
1
mu chun v ca cc mu th y = 0.295x + 2.481
0.8 2
R = 0.996
Mu v chun Ct(S35) Ct(lectin) DCt 0.6

Chun GMO 0.0% ND 24.40 ND 0.4

0.2
Chun GMO 0.1% 35.96 24.30 11.66 DCt
0
Chun GMO 0.5% 33.19 23.60 09.59 -0.2 0 2 4 6 8 10 12 14

Chun GMO 1.0% 32.12 23.60 08.52 -0.4

-0.6
Chun GMO 2.0% 31.34 24.00 07.34
-0.8
Chun GMO 5.0% 29.76 23.80 05.96
-1
Mu 1 27.22 21.85 05.37 -1.2

Mu 2 ND 20.73 ND Biu 10: Biu chun mi quan h gia DCt

v t l GMO

T hm s tng quan y = -0.295x + 1.592 vi y l log(GMO%) v x l DCt,

chng ta s tnh c mu 1 vi DCt l 05.37 th t l GMO% l 7.9%, cn ca mu 2
l 0-0.1%.

3. Pht hin khc bit SNP v xc nh kiu di truyn

Sn phm khuch i ca cc trnh t ch c th khc bit ch mt nucleotide, gi

l SNP, hay l c th khc bit mt s nucleotide ca mt trnh t. Pht hin cc khc
bit ny nhiu lc rt c ngha, chn hn pht hin t bin khng thuc l pht hin
cc SNP, hay phn bit cc kiu di truyn ca cc tc nhn ch c trong mu th l
cc bnh phm ly t bnh nhn l pht hin cc trnh t khc bit. Real-time PCR
hon ton c th ng dng pht hin khc bit SNP v xc nh kiu di truyn.

Vi real-time PCR dng mu hunh quang chn lm cht pht hunh quang th
phi s dng mu HRM v phi thc hin chng trnh phn tch HRM sau khi hon
tt chng trnh lun nhit th mi c th pht hin c khc bit SNP v xc nh
c kiu di truyn (xem phn k thut phn tch phn gii cao nhit chy).

Vi real-time PCR dng probe lm cht pht hunh quang th ty s tin li hay
quen thuc vi k thut no m nh nghin cu hay ngi lm th nghim c th chn
phng php thch hp vi mnh. V d vi Taqman probe, pht hin s khc bit
SNP v nh genotype, nh nghin cu phi thit k cc Taqman probe (mang cc cht
pht hunh quang R khc nhau) bt cp trn cng v tr ca sn phm khuch i
nhng cc probe ny c s khc bit v trnh t tng ng vi s khc bit SNP hay
khc bit trnh t gip phn bit genotype. Trong giai on bt cp th probe c trnh t

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tng thch nht vi trnh t ch trn sn phm khuch i s bt cp ln si ch v
s b Taq polymerase thy gii, do vy ng biu din khuch i tng ng vi mu
hunh quang ca probe s ngc ln v n cc i trong khi tn hiu ca cc mu
hunh quang khc s vn nm di hunh quang nn v cc probe tng ng vn cn
nguyn vn v khng b thy gii. Hnh 29 di y minh ha c ch hot ng ca
Taqman probe trong pht hin SNP hay genotype.

R2 Q2

R1 Q1

R2 Q2

R1

Q1

R1 Q1

R2 Q2

R1 R2

R2

Q2

R1

Q1

R2

Q2

Hnh 29: Minh ha c ch hat ng ca Taqman probe trong pht hin khc bit SNP hay genotype
vi probe R1 cho wild type v probe R2 cho kiu t bin. Hnh trn ch c wild type. Hnh
gia ch c t bin, v hnh di l heterozygote va wild type, va t bin

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 Phng php real-time PCR dng probe lai (FRET) cng c cc nh nghin cu
s dng pht hin cc khc bit SNP v c bit l khc bit genotype. Phng php
ny t ra u im hn phng php s dng Taqman probe trong pht hin cc khc
bit genotype, nht l khi cc genotype ca tc nhn ch ngoi khc bit nhau v trnh
t c trng cn cha cc khc bit trnh t do s khc bit dng hay khc bit v t
bin trong qu trnh pht trin (v d HIV, HCV, HBV...). L do l rt kh thit k
c Taqman probe c hiu cho mt genotype v ngoi trnh t c hiu cho genotype
Taqman probe s phi cha cc trnh t bt cp vi cc trnh t bin i v chnh iu
ny s lm cho hiu qu bt cp ca Taqman probe c hiu cho genotype s khng
cao, do vy kt qu pht hin genotype s khng c hiu nh mong mun. Trong khi
thit k probe lai s d dng hn, ch cn quan tm n trnh t khc bit cho
genotype m khng nht thit phi quan tm n cc bin i trnh t khc, v cho d
c mang cc trnh t bin i ngoi trnh t c hiu genotype th khi phn tch nh
chy bng chng trnh HRM th mi genotype s biu hin mt nh chy c trng
ph thuc ch yu vo trnh t c hiu ca genotype m hai probe lai bt cp vo
(xem phn probe lai). Biu 11 l mt v d ca ng dng probe lai trong pht hin
genotype ca HCV bng real-time PCR c hiu vng 5-NC.

Biu 11: Kiu nh chy tng ng vi genotype HCV trong kt qu phn tch nh chy sn phm
khuch i ca real-time c hiu vng 5NC v dng FRET probe (trch t Clinical Chemistry
48:12)

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