Nhng vn v k thut cn c quan tm khi p dng

PCR v real-time PCR trong phng th nghim chn on

S khc bit gia phng th nghim chn on v phng th nghim nghin cu

Phng th nghim nghin cu l phng th nghim m cc th nghim c thc hin

p ng mt nhu cu nghin cu no nhm gip nh nghin cu gii p mt vn
ca khoa hc. Kt qu ca mt phng th nghim nghin cu khng lin quan trc tip
v tc thi n quyt nh ca ngi gi mu xt nghim, do vy trong chn nui hay
thy sn s khng lin quan n quyt nh hy hay c lp mt qun th gia sc hay tm
c, hay trong y hc khng lin quan n quyt nh iu tr ca bc s trn bnh nhn. Do
vy kt qu ca phng th nghim nghin cu s khng cn thit phi kp thi n tay
ngi gi mu xt nghim.

Phng th nghim chn on l phng th nghim c nhim v chnh yu l lm xt

nghim trn cc mu th pht hin c tc nhn gy bnh v tr li kt qu n
ngi gi yu cu xt nghim h th cho cc cch gii quyt c th trn cc vt ch
c ly mu gi i xt nghim. Trong chn nui hay thy sn, kt qu ca mt phng
th nghim chn on s rt quan trng v c lin quan n quyt nh ca gii hu trch
tiu hy hay c lp mt by n gia sc hay tm c. Trong y hc, phng th nghim
chn on cn c gi l phng th nghim lm sng v kt qu xt nghim s c cc
nh lm sng tham kho a ra pht iu tr trn bnh nhn. Chnh v vy mt
phng th nghim chn on phi l phng th nghim khng ch cho kt qu chnh xc
m cn phi cho kt qu kp thi n tay ngi lm xt nghim.

Vic ng dng PCR v real-time PCR trong chn on tr nn ngy cng d dng v
y l mt cng ngh m, phng th nghim c th t pha ch cc thuc th thc hin
th nghim. Ngoi ra, nhu cu s dng PCR v real-time PCR trong chn on ngy cng
nhiu v c nhiu tc nhn gy bnh ch c th chn on hay theo di c bng cng
ngh PCR v real-time PCR. Khng ch vy, hin nay trn th gii, cng ngh PCR chn
on sp ht bn quyn nn ang c s bng n cc sn phm chn on da trn cng
ngh PCR v real-time PCR c nhiu hng sn xut cung cp. Do vy, c th ni PCR
hin nay khng cn b hp trong cnh ca ca cc phng th nghim nghin cu m
dn dn xm nhp vo trong cc phng th nghim chn on.

115
 Tuy nhin vic p dng PCR v real-time PCR trong chn on cng i hi ngi lm
th nghim phi bit quan tm n cc vn k thut c lin quan kt qu xt nghim
t c chnh xc cao, v t c nhy cm tuyt vi ng nh bn cht ca
PCR v real-time PCR, v phi kp thi n tay ngi ch nh xt nghim.

Cc vn k thut cn quan tm p dng PCR v real-time PCR trong chn on

1. Tin trnh xt nghim PCR v real-time PCR

K t khi ly mu th gi n phng th nghim lm xt nghim PCR hay real-

time PCR chn on cho n khi kt qu xt nghim n tay ngi cho ch nh xt
nghim, tin trnh s qua cc bc m trong mi bc u c nhng vn k thut rt
cn c quan tm bi ngi lm xt nghim, ngi ly mu xt nghim, v ngi
nhn s dng kt qu xt nghim ny. S 3 di y trnh by cc bc trong
mt tin trnh xt nghim.

Ly v chuyn mu th i lm Cc vn k thut trong

v loi tr ngoi nhim sn phm khuch i

xt nghim PCR/realtime PCR ly v gi mu th

Vn ngn nga nhim cho

Chun b mu th v tch chit Cc vn k thut trong
nucleic acid t mu th tch chit nucleic acid (NA)

Thc hin phn ng khuch i Cc vn k thut trong

nucleic acid (PCR/real-time PCR) khuch i NA

Lm th nghim c kt qu Cc vn k thut trong th nghim

Phn tch kt qu c KQ v trong phn tch KQ

S dng kt qu xt nghim Cc vn k thut trong

PCR/real-time PCR s dng kt qu

S 3: Tin trnh mt xt nghim PCR/real-time PCR chn on v cc vn k thut cn c quan tm

116
2. Cc vn k thut cn quan tm

a. Cc vn k thut cn quan tm khi ly v gi mu th

Mu th c ly lm xt nghim PCR/real-time PCR l cc mu c ly t vt

ch ti ni m ngi ly mu cho l c th c s hin din ca nucleic acid ca tc
nhn ch, do vy phi ly mu th ng ch. V d pht hin M. tuberculosis gy
lao phi trn bnh nhn th mu th phi ly l m hay dch ht t ra kh/ph qun,
khng th ly mu hay huyt thanh. Hay mun pht hin Cytomegalovirus trong bnh
nhn th mu th tt nht l mu tan phn tch huyt tng hay tch bch cu,
khng th l mu ng tch huyt thanh c.

Vt liu dng ly v gi mu th cng phi c quan tm v nu khng, cc

nucleic acid ca tc nhn ch c trong mu s b phn hy hay mu th s cha cc
cht c ch phn ng khuch i sau ny. Do vy vt liu tt nht ly v gi mu
th l cc vt liu tinh sch v mt sinh hc, ngha l khng nhim cc protein hay cc
nuclease tit ra t cc vi sinh vt nh endonuclease, ribonuclease; v ch s dng mt
ln, khng nn c ra xi li. Hnh 59 m t mt s vt liu nn c s dng
ly v gi cc mu th lm xt nghim PCR/qPCR. Cc vt liu ny hin c sn ti
cng ty Nam Khoa.

Hnh 59: Cc l v tube nh th ny nn c s dng ly v gi cc mu th lm xt nghim PCR/qPCR

bo qun v chuyn ch mu th vn k thut cn quan tm l lm th no

mu gi nguyn trng, nucleic acid ca tc nhn ch khng b phn hy trong qu
trnh bo qun. Do vy i vi cc mu m nucleic acid ch cn pht hin l DNA th
c th bo qun ngn ngy (trong vng 48 gi) t mt 2-8oC, cn nu bo qun lu
di th cn gi -18oC n -30oC. i vi RNA, nu l cc mu t hay khng b tp
nhim nh mu, dch c th th c th bo qun 48 gi 2-8oC, nhng i vi cc mu
117
tp nhim nhiu nh qut hng, qut mi sau tm virus, m...hay cn bo qun di
ngy th phi gi mu -70oC. chuyn ch mu th ch cn chuyn ch trong thng
lnh vi thi gian chuyn ch khng qu 48 gi. Nu thi gian chuyn ch lu hn th
rt cn phi gi trong kh.

Ni chung, cc vn k thut c nu ln l ch nhm mt mc tiu, l ly

mu cho ng ch l ni c s hin din ca nucleic acid ch mun tm, phi gi v
chuyn ch mu th n phng th nghim trong iu kin sao cho nucleic acid mun
tm khng b phn hy cng nh mu th khng b nhim cc cht sinh hc gy c ch
phn ng khuch i v mu th khng bi nhim cho.

b. Cc vn k thut trong chun b mu th v tch chit nucleic acid

Trong khu ny, mc tiu chnh l phi lm sao tch chit c ti a nucleic acid
ch d lng nucleic acid ca tc nhn ch c nh v c b pha long qu thp trong
mu th. Tch chit thu c phi tinh khit, khng ln cc protein hay cht gy c
ch phn ng khuch i.

Ty loi mu th v cng ty loi tc nhn ch c trong mu th m c nhng

mu th c th a vo tch chit nucleic acid ngay, nhng cng c nhng mu th
cn phi c chun b trc khi a vo tch chit nueic acid (NA). V d cc mu
th nh huyt thanh, huyt tng, cy t bo...vi cc tc nhn ch nh virus hay cc
vi khun c cu to t bo khng c bit th c th a vo tch chit nuleic acid ngay,
nhng vi cc mu th nh mu m, mu sinh thit hay cc vi khun c cu to t bo
c bit nh M. tuberculosis, nm men, nm si, m thc vt nh cy-l-r-hoa th cn
phi c a vo chun b trc cc cu trc ca m tan i (vd phi s l
proteinase K), t bo v ra (vd dng cc cht ty TRITON X100, hay tn siu m,
hay nghin trong nitrogen lng...), hay nh trong trng hp cc lt ct sinh thit c
sp th cn phi loi b sp khi m, hoc cc mu c cc cht nhy nh m th cn
phi lm tan nhy (bng NALC...) trc khi a vo tch chit nucleic acid...Do vy,
ngi lm xt nghim cn phi bit khng ch cc mu th no cn phi qua giai an
x l mu trc khi tch chit NA m cn phi bit phi s dng phng php g vi
qui trnh k thut nh th no.

118
 Ty loi mu th; ty loi tc nhn ch l vi khun, virus, vi nm, hay t bo c
nhn; v ty NA phi tch chit l DNA hay RNA m phi la chn phng php tch
chit cho ph hp, khng nn ln ln. V d phng php Boom rt tt tch chit
cc dch sinh hc hay cc mu th t ln DNA ca vt ch v nguyn tc l da vo s
hp ph ca DNA trn ht silica, nn nu mu c qu nhiu DNA vt ch nh cc m
sinh thit th DNA tc nhn ch s b cnh tranh khng bm c vo ht silica. Do
vy, tch chit DNA cho cc mu th l m v sinh thit th nn chn phng php
phenol/chloroform hn l phng php BOOM. Cng khng th s dng phng php
tch chit cho RNA vo tch chit DNA, do vy m chng ta thy phng php
BOOM rt tt cho tch chit DNA nhng li rt km cho tch chit RNA. Khi la chn
phng php hay b thuc th tch chit cho tc nhn virus hay vi khun ch, cng
phi xem k b tch chit hay phng php tch chit NA m mnh ang la chn c
p dng c cho vi khun hay virus khng? V c khi s tht bi trong xt nghim
PCR l do nhng s v ny.

Tch chit NA l mt khu rt quan trng; tch chit NA phi m bo c

nhy th xt nghim PCR mi c th t c nhy mong mun. Do vy khi t pha
mt b thuc th tch chit NA hay khi sn xut mt b thuc th tch chic NA,
trc khi a vo s dng trong xt nghim PCR/real-time PCR, phng th nghim hay
nh sn xut phi kim tra cht lng xem b tch chit c m bo c nhy
hay khng. Phng php kim tra l phi th trn cc pha lang ca mt tc nhn
ch (l i tng m phng th nghim hay ngi dng s phi s dng b tch chit
NA thc hin xt nghim) hay ca mt tc nhn khc tng ng. t nhy l
b tch chit NA khi th nghim trn cc pha long tc nhn ch ny phi t c
gii hn pht hin (limit of detection, LOD), tc l t c s lng vi khun ch
thp nht c trong mt n v th tch mu th m phng php hay b th nghim c
th tch chit v pht hin c.

Minh ha di y (hnh 60) l mt kt qu kim tra cht lng b kit tch chit
NK
DNA tn l DNAPREP-BOOM do cng ty Nam Khoa sn xut v c kim tra
cht lng. Phng php kim tra l s dng cc pha long ca vi khun M.
tuberculosis, cho vo tch chit DNA vi b thuc th sn xut ng thi so snh
NK
vi b thuc th DNAPREP-BOOM c sn xut trc . Kt qu kim tra cho

119
 NK NK
thy b thuc th DNAPREP-BOOM mi sn xut v b thuc th DNAPREP-
BOOM sn xut trc u t c kh nng tch chit vi khun ch n pha
lang 10-4, cha 1 vi khun trong mt th tch 100ml dch vi khun a vo tch chit.
Trong phng php ny nu huyn dch vi khun c b gim cht lng th kt qu
NK
kim tra vn c th c c nh c so snh DNAPREP-BOOM mi sn xut vi
NK
DNAPREP-BOOM sn xut l trc v l ny t cht lng.

Hnh 60: Kt qu kim tra cht lng b thuc th NKDNAPREP-BOOM do cng ty Nam Khoa sn xut. i
tng dng kim tra b thuc th l huyn dch vi khun M. tuberculosis TB1 cha 10.000 t bo vi
khun/ml c pha long theo h s 10 c 1,000 t bo vi khun/ml (T2), 100/ml (T3), 10/ml (T4), v
1/ml (T5). Phng php kim tra l s dng b thuc th NKDNAPREP-BOOM cn kim tra v b
NK
DNAPREP-BOOM c sn xut trc thc hin tch chit trn cc huyn dch vi khun pha
long ny, th tch huyn dch tch chit l 100ml, ri ly 10ml dch tch chit chy PCR pht hin
M. tuberculosis. Pht hin DNA ca M. tuberculosis c trong dch tach chit qua in di pht hin sn
NK
phm khuch i 249bps. Kt qu cho thy c hai b thuc th DNAPREP-BOOM mi v c u t
LOD l 1 t bo vi khun M. tuberculosis trong th tch tch chit.

Khng ch kim tra cht lng cc b thuc th tch chit NA c pha ch trc
khi s dng, m ngay c trong mi ln s dng lm xt nghim, b thuc th tch
chit cng phi c kim sot nhy bng cch cho nucleic acid chng ni ti
nng LOD vo mu chng [-] v nu mun, c th cho vo mu th c thc
hin tch chit cng lc vi cc mu th (xem phn chng ni ti).

c. Cc vn k thut trong khuch i nucleic acid ch

V nguyn tc th PCR/real-time PCR t c nhy rt cao, tuy nhin trn

thc t th nhy cn ty thuc rt nhiu vo s thit k hay chn la mi; vo thit
b lun nhit; vo ha cht, c bit l enzyme Taq polymerase c s dng. Khu

120
khuch i s rt kh t c nhy v duy tr nhy trong qu trnh lu tr nu
nh sn xut hay ngi lm th nghim khng chn lc Taq polymerase s dng.

Ngoi cc yu t k thut cn phi quan tm trn, m bo c nhy ca

khu khuch i, nu t pha hay sn xut PCR mix cho phn ng khuch i, ngi
lm th nghim hay nh sn xut phi kim tra nhy ca cc PCR mix trc khi a
vo s dng. Phng php kim tra l th nghim trn cc pha lang ca DNA ch
bit trc s lng copy trong mt n v th tch. PCR mix c gi l t
nhy khi t c gii hn pht hin (LOD) l mt copy DNA ch cho vo PCR mix,
ngha l trong th tch mu cho vo PCR mix ch cn c 1 copy DNA ch l c th cho
kt qu khuch i thnh nhiu bn sao c th pht hin c khi phn tch kt qu
(xem mc kim tra nhy ca PCR mix pha bi PCR-cc vn c bn).

10

100

1,000

10,000

Biu 16: Kt qu kim tra cht lng real-time PCR mix s dng mi v Taqman probe nh lng HBV-
DNA. Kt qu kim tra cho thy nhy ca PCR mix t LOD 1 copy HBV-DNA trong mt th
tch mu cho vo PCR mix. Cc ng biu din mu xanh sng l ng biu din khuch i
ca chng ni ti c cho vo PCR mix nng LOD cng vi mu kim tra.

Biu 16 minh ha mt kt qu kim tra cht lng real-time PCR mix dng
mi v Taqman probe pht hin v nh lng HBV-DNA do cng ty Nam Khoa
sn xut. Kt qu kim tra cho thy nhy ca PCR mix t LOD l 1 copy cho mt
th tch mu cho vo PCR mix, y chnh l nhy i hi ca PCR/real-time PCR
chn on.
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 Ngoi vic kim tra cht lng cc PCR mix trc khi a vo s dng, ngi
lm xt nghim khi thc hin xt nghim PCR cng phi kim sot cho c nhy
ca PCR mix c s dng trong mi ln lm xt nghim. Thc hin vn kim sot
ny l mt iu kin bt buc v d PCR mix c c kim tra t cht lng sau khi
pha ch hay sn xut, vn c th gim cht lng trong qu trnh lu tr hay bo qun,
c bit khi c pha ch bng ngun Taq polymerase khng m bo cht lng.

C nhiu cch kim sot cht lng PCR mix mi khi lm xt nghim
PCR/real-time PCR chn on. C th s dng chng [+] l DNA ch c pha sn
hay c nh sn xut cung cp nng LOD cho vo PCR mix trong mi ln lm
xt nghim PCR/real-time PCR chn on, hay c th s dng DNA chng ni ti s
dng chung mi c pha nng LOD cho vo PCR chung vi tch chit DNA t
mu kim sot c ch PCR v ng thi kim sot nhy ca PCR mix (xin xem
thm cc mc chng ni ti, chng dng, v chng m trong phn cc chun mc
v kim sot cht lng cho xt nghim PCR/real-time PCR dnh cho chn on).

d. Nga nhim cho v loi tr ngoi nhim sn phm khuch i

Mun s dng PCR/real-time PCR trong chn on, ngi lm xt nghim phi
nhn din c c cc bin php k thut ngn nga hay loi tr cc nguy c ngoi
nhim lm cho mu th khng c tc nhn ch li b c kt qu dng tnh khi lm xt
nghim, ngha l b nguy c dng tnh gi. Ngoi nhim trong xt nghim PCR/real-
time PCR chn on c th xy ra do hai nguyn do.

Nguyn do th nht l nhim cho lm cho mu m hay cc thuc th lm xt

nghim b nhim nucleic acid ch t cc mu dng. Vi nguyn do ny th ngi
lm th nghim han tan c th phng nga c bng cc b tr phng th nghim
mt cch hp l vi cc phng tin v thit b ph hp, v d thit k khu vc pha
cc thuc th cch xa khu lm mu; khng ln ln cc thuc th pha v ang
dng vi cc nguyn liu v dung dch s c dng pha thuc th; cc dng c v
ti liu dng trong khu vc lm mu khng c em qua khu vc pha thuc th;
khng phng th nghim vi sinh nui cy tc nhn ch trong phng th nghim
PCR/real-time PCR chn on pht hin ng tc nhn . Ngoi cch b tr phng th
nghim lm vic cho hp l, ngi lm xt nghim PCR/real-time PCR phi c

122
hun luyn nhun nhuyn cc thao tc v trng c bit trong ng m tube, chai l,
thao tc pipetting, trong b tr cc vt liu v dng c khi thao tc...

i vi nguyn do th hai l ngoi nhim sn phm khuch i th ch vi cc gii

php k thut nh nu trn vn kh c th trnh c nguy c ny, l do l trong
mt phng xt nghim PCR/real-time PCR chn on, sn phm PCR s tch t ngy
cng nhiu v chc chn khng sm th mun cc sn phm PCR ny s nhim vo cc
dng c, thuc th, thit b...v n mt lc no ngi lm xt nghim s nhn dng
c thm ha ny khi cc mu xt nghim PCR u cho kt qu dng tnh. n lc
ny th ch c mt gii php l b tan b thuc th, kh nhim tan b phng th
nghim, ng ca vi thng...Ri li m ca lm li, ri thm ha ti xut hin...M t
nh vy chng ta thy rng khng th c bin php no hiu qu nga ngoi
nhim sn phm khuch i, m ch c mt cch duy nht l phi loi tr c nguy
c ny. lm c iu ny th cch duy nht l phng th nghim PCR chn on
ngay t u phi s dng cc PCR mix c pha thm dUTP v UNG. C ch loi tr
c ngoi nhim sn phm khuch i bng dUTP v UNG c ni chi tit trong
mc loi tr ngoi nhim sn phm khuch i ca bi PCR-cc vn c bn.

Qua phn tch nh trn, chng ta thy l nu mun lm c xt nghim PCR

chn on, khng th khng s dng bin php dng PCR mix c pha dUTP v UNG.
Nu khng p dng bin php ny, khng sm th mun, thm ha ngoi nhim sn
phm khuch i s xy ra. Tuy nhin cng c cc vn k thut khi dng dUTP v
UNG v nu khng chn c enzyme UNG hiu qu v ph hp th nhy ca
PCR/real-time PCR s gim v/hay khng th c hiu qu loi tr ngoi nhim sn
phm khuch i. Chnh v vy ngi s dng phi kim tra nhy v kim tra kh
nng loi tr ngoi nhim sn phm khuch i ca PCR mix c cng b l c
dUTP v UNG. Hin nay tt c cc hng nc ngoi sn xut cc PCR mix dng cho
PCR/real-time PCR chn on u s dng dUTP v UNG. Ti Vit Nam, cng ty
Nam Khoa l cng ty tin phong v hng u p dng gii php ny. Tt c cc b
thuc th PCR/real-time PCR dng trong chn on do cng ty Nam Khoa sn xut
u s dng PCR mix c dUTP v UNG bng ngun ha cht chn lc m bo
khng gim nhy m hiu qu loi tr ngoi nhim sn phm khuch i vn cao
vi bng chng kim tra cht lng v nhy lun i km vi sn phm.

123
e. Cc vn k thut trong th nghim in di trn thch c kt qu

i vi phng php PCR c in th mun c c kt qu, ngi lm th

nghim phi lm tip th nghim xc nh c trong ng phn ng c sn phm
khuch i c hiu hay khng. Phng php thng c p dng nht trong cc
phng th nghim PCR chn on l dng k thut in di xc nh kch thc sn
phm khuch i c ng nh kch thc mong mun hay khng. Chnh v y l
phng php thng c s dng nht, nn chng ti xin a ra y mt s vn k
thut cn c ngi lm th nghim quan tm.

Chn t l % agarose in di cho ph hp vi kch thc sn phm khuch

i. Trung bnh nht l dng t l 2%. i vi cc sn phm khuch i ln trn
500bps th nn dng agarose 1.5%, cn i vi cc sn phm khuch i kch
thc di 100bps th nn dng agarose trn 2.5%.

Khi pha gel agarose, lu l phi lm tan han tan agarose trong m in di,
v lm c iu ny phi cho agarose vo bnh c sn mt t dung dch
m din di, nh vy trnh agarose b bm vo y bnh ri s khng tan c sau
khi un. Trc khi cho vo l vi sng phi lc trn agarose ha u trong dung
dch m. Khi un, phi t nht hai ln ly bnh ra khi l vi sng lc trn u
ri mi cho vo un tip. Phi m bo agarose tan u trong dung dch m,
khng cn thy ln cn d rt nh. C nh vy mi c kt qu in di tt.

Trc khi agarose vo khun, phi m bo khun nm trn mt phng nm

ngang kim tra bng bng kh b dy ca agarose tht s dy u nh nhau v
kt qu in di s chnh xc hn

Trong PCR chn on, khng nn trn m ti vi sn phm PCR trn giy
paraffin v lm nh vy s gy nguy c ngoi nhim. Nn cho m ti vi th tch
bng th tch ca dung dch phn ng c trong tube PCR ri trn ngay trong
tube.

Th tch mu cho vo ging khng nn qu t m phi t 15ml tr ln v c nh

vy mi c th pht hin c cc kt qu [+] yu

Lun in di km vi thang DNA v y l mt chun khng ch dng xc

nh kch thc ca cc vch in di trong cc mu m cn l mt chun kim
124
 sot cht lng ca in di. Gi l cht lng din di tt khi thang DNA tch c
nhau r rng v tt c cc vch ca thang.

Trong mt kt qu in di chun mc cho PCR chn on cn cn phi c mt

ging cho chng [+] so snh kch thc ca sn phm khuch i trong vch
in di t cc ging cha mu, v mt ging cho chng m chng minh qu
trnh lm xt nghim PCR chn on khng b ngoi nhim (xem chun mc cho
PCR/real-time PCR chn on). Hnh 61 minh ha mt kt qu chun mc ca
xt nghim PCR c qua in di, phn tch trn hnh chng ta s thy lun c
chng [+], chng [-] v thang DNA.

MK Thang DNA
1 Chng [+]
2 WSSV [+]
MK 6 5 4 3 2 1
3, 4 WSSV [+], IC [+]
5 WSSV [-], IC [+]
6 Chng [ -]
633bps (WSSVout+)
#260bps (WSSVout/in+)
221bps (WSSVin+)
195bps (IC+)

Hnh 61: Minh ha mt kt qu nonstop nested PCR pht hin WSSV c thc hin ti cng ty Nam Khoa.
Lu kt qu din di phi lun lun c cc chng [+], chng [-], v phi c thang DNA

Phi thay dung dch m din di trong mng in di trong vng khng qu 1
tun m bo cht lng in di lun tt.

Nu khng mun tip xc vi ethidium bromide, mt ha cht c tim nng

gy ung th mnh, th c th s dng cc mu hynh quang chn khc t c hi
hn nh SYBR (SYBR safe ca Invitrogene), hay han tan khng c hi nh
GelRed hay GelGreen (ca Biotium). Tuy nhin gii php ny s mc tin hn rt
nhiu.

Nu phi s dng ethidium bromide th phi tuyt i ch cc bin php an

tan. Cc bin php an tan khi s dng ethidium bromide c trnh by tip
theo y.

125
f. Bin php an tan khi s dng ethidium bromide

Ti sao phi thao tc vi ethidium bromide?

Ethidium bromide (EB) l mt loi mu chn (intercalating dye) c c im chn

vo cc si i DNA v lm cc si i ny pht hunh quang c di nh sng cc
tm. Da vo c im c bn ny ca EB m trong sinh hc phn t, ngi thc hin
th nghim thng dng EB nhum cc bng in di (cc vch DNA tch bit nhau
da vo kch thc sau khi in di trn thch hay trn gel polyacrylamide) v pht hin
cc bng ny qua hp n UV trn mt knh lc.

c th nhum cc bng DNA trn thch in di, ngi ta c th cho EB pha

vo trong thch khi thch hay nhum thch sau in di bng cch ngm thch in
di vo chu nc c pha EB. Tu thuc vo s chn la u im no m ngi lm
th nghim c th chn cch pha EB vo thch hay cc nhum EB sau khi in di.
Cch pha EB vo thch th s cho php c kt qu ngay sau khi in di nhng li lm
cho ton b h thng mng-khay-lt in di u b nhim EB. Cch nhum EB sau
in di th gi c h thng mng-khay-lt in di khng b nhim EB nhng phi
ch n 30 pht sau khi in di nhum EB ri mi c c kt qu.

Thao tc an tan vi Ethidium bromide

V c im chnh ca EB chn vo cc si i DNA nn EB c xem v

c chng minh l mt cht sinh ung th mnh. Do vy ngi s dng EB lm th
nghim phi tun theo cc qui tc an ton trnh nguy c tip xc trc tip vi EB.
Sau y l mt s qui tc an ton nn tun th:

Ph n ang c thai, cho con b, hay ang d tnh mang thai khng nn lm
vic trong phng th nghim c s dng EB.

Trong phng th nghim phi c hai thng cha cht thi c nhim EB: mt
thng dnh cho cht lng v thch, mt thng dnh cho cht rn nh gng tay,
giy lau, nha. Ti cc nc tin tin, hai thng cht thi ny phi c gi i
hu b mt cch c bit. Ti Vit Nam, chng ta cha c mt c quan no ng
ra x l cc loai cht thi c bit nguy hi nh EB nn tm thi c th x l nh
sau trc khi cho vo rc y t: l trc khi cho cht thi vo thng, phi lt
thng vi bao nylon dy cht thi c cho vo bao nylon ch khng tip xc
126
trc tip vi thng, ngm cht thi vi cloramin P 10% (cht thi lng hay thch)
hay rc trc tip bt choramin P (cht thi rn) v gi t nht 1 thng trc EB
b cloramin P phn hu trc khi giao qua x l rc y t.

Trnh tuyt i nguy c tip xc trc tip EB qua cc bin php nh sau:

(1) Khi cn hay pha EB th phi mang gng tay, khu trang, v eo knh bo
v. Sau khi cn th bt c cc dng c no tip xc vi EB k c gng tay, khu
trang u phi cho vo thng cha cht thi rn. Nn dng que xc ho cht bng
nha xc EB khi cn v sau b que xc ny i cng vi gng tay ch khng
nn dng que xc ho cht bng inox v chng ta khng th m bo lau sch EB
sau khi cn.

(2) Nu pha EB vo trong thch th phi dng mt chai un thch khng nhim
EB v sau khi un tan hon ton thch, chng ta cho thch vo mt chai th hai
l chai thng dng trn EB vo thch. Ch thch trong chai th 2 ny ngui
n 50oC ri mi cho EB vo (thng mi 50ml thch, ch cho 1-2l EB
10mg/ml), trnh cho EB vo thch khi thch cn nng v nguy c EB bc ln cng
vi hi nc khi thch cn nng. Sau khi thch vo mng, chai cha thch c
nhim EB phi c sc bng nc lm sch thch v nc sc chai ny phi
c cho vo thng cha cht thi EB lng. Phi eo gng, khu trang v knh bo
v khi thao tc, v sau khi thao tc, phi b gng tay v khu trang cng nh u
pipette ht EB vo thng cha thi EB rn. Micropipette dng ht EB l ch
c chuyn dng ht EB, phi c ghi ch pipette nhim EB v c gi
ring trong mt hp c ghi ch hp cha nhim EB. Tt nht thao tc trn EB
vo thch v EB vo trong mng in di phi c thc hin trong mt hood
pha ho cht c c p sut m v kh thi qua lc hp ph ho cht trc khi a
ra ngoi (biosafety class I). Nu khng c t ny th nn t ng mt t thch
trn np t l my ht lc than hot tnh (xem hnh 62) v lc ny phi c thay
mi 4-6 thng mt ln. Sau khi thch c hon ton, nu cha s dng th c th
gi thch nguyn trong khun ri cho vo bao nylon dy c mt t dung dch m
in di ri hn hay ct ming li. Thao tc cho thch vo bao nylon phi tht s
thn trng trnh mt ngoi bao b nhim EB. Bao nylon cha thch phi cho
vo gi trong t mt 2-8oC ti mt ngn hay hp dnh ring cho n khi s dng.

127
Bao nylon cha thch sau khi dng xong phi cho vo thng cha thi rn. Thch
sau khi in di nu cha dng ht cc ging th khng ly khi mng in di m
nn li ngay trong mng in di cho n khi dng ht (khng nn qu 1 tun).
Thch in di sau khi dng ht th phi cho vo thng cha thi EB lng. Dung
dch m in di cng vy, phi thay sau mi tun v dung dch m b i phi
vo thng thi EB lng, khng c vo chu ra trong phng th nghim.
Mng-khun-lt in di sau khi dng nn ngm ra trong mt chu nha ring
c ghi ch chu ra nhim EB v dch ra ny phi c vo thng cha thi
EB lng. Phi c mt hp cha ring gi cc mng-khun-lt nhim EB v
phi ghi ch hp cha nhim EB. Phi mang gng tay khi s n cc mng hay
hp cha nhim EB ny, v gng tay sau khi dng phi cho vo thng cha thi
EB rn.

(3) Nu nhum EB sau in di th cc thao tc thch, bo qun thch s

khng b nhim EB nn chng ta khng lo v an ton cc khu ny. Nhng khi
thao tc pha thuc nhum, nhum v sau nhum th chng ta phi ch cc bin
php an ton: Trc ht phi dng mt khay nhum ring bit v c ghi r l
khay nhum nhim EB. Phi mang gng tay, khu trang v knh bo v khi pha
dung dch nhum, thao tc nhum v sau nhum. Gng tay, khu trang v cc u
pipette dng ht EB phi
c cho vo thng cha
thi rn EB sau khi dng
xong. Micropipette dng
ht EB l ch c
chuyn dng ht EB,
phi c ghi ch pipette
nhim EB v c gi
ring trong mt hp c ghi
ch hp cha nhim EB.
Phi mang gng tay khi s
n cc vt liu c ghi ch Hnh 62: M hnh mt t dng gel in di t ng. T ny
hin ang c cng ty Nam Khoa sn xut v cung cp
nhim EB ny, v gng n cc phng th nghim PCR khi c yu cu.

128
 tay sau khi dng phi cho vo thng cha thi EB rn. Dung dch nhum sau khi
dng xong, phi vo thng cha thi EB lng.

(4) Phi c bit quan tm n an ton khi thao tc quan st thch in di trn
hp n UV v y l khu m ngi lm th nghim d b nguy c tip xc trc
tip vi EB nht. Sau y l cc khu cn lu :

(i) Phi mang gng tay khi ly thch sau khi in di mang n hp n UV, v
trnh ri vi cc git dung dch c EB t thch xung sn nh, ngi thao tc
phi dng mt giy thm hng bn di khi mang thch n hp n UV v giy
thm ny phi cho vo thng cha thi rn nhim EB. Tt nht cho thch ln mt
khay c ghi ch khay nhim EB mang n quan st trn hp n UV.

(ii) Sau khi t thch ln hp n UV, phi dng ngn tay cha chm g n
EB bt cng tc m v tt n UV cng tc ny khng b nhim EB. Nu
dng h thng geldoc ca Biorad th cc thao tc m ca, bt tt cng tc hay
nhn cc nt chnh trn geldoc hay trn bn phm vi tnh phi c thao tc bng
mt bn tay khng nhim EB.

(iii) Hp n UV sau khi dng xong phi c lau sch bng giy thm nuc
v cc giy lau ny phi c cho vo thng cha thi rn nhim EB.

(iv) Hp n UV l ni nhim EB rt cao, do vy trnh s hay chm vo hp

n UV vi tay trn.

(v) Mt lu cn c lun tuyt i tun theo: trnh bn tay mang gng

nhim EB dng n bt c vt dng g trong phng th nghim, c bit cc ni
rt d b s vo bi tay trn nh: Micropipette; tay nm m ca t lnh, ngn ko;
tay nm ca, bt vit...

Trn l cc thao tc an ton trong phng th nghim c thao tc vi EB m phng

th nghim nn t chc v nhn vin nn tun theo. Nn nh l nhim EB vo c th
khng cho tc hi tc th m c th gy hu qu sau nhiu nm. Do vy cn tc v
y ny, tuy nhin cng khng nn qu cc oan v phng i cc mi nguy c th
phng nga c ny ty chay khng dng EB v y l mt ho cht hu hiu nht
v r tin nht phn tch c kt qu sau in di.

129
g. Vai tr ca chng [+], chng [-] v chng ni ti

Chng [+] cho PCR mix

nh ngha: l DNA c cho vo PCR mix cho sn phm khuch i c

kch thc v trnh t ng ging ht sn phm khuch i ca DNA ch tch chit
c t bnh phm. Thng thng chng [+] cho PCR mix l dung dch pha lang
nng bit trc ca plasmid c chn an DNA ch.

Vai tr: Chng [+] cho PCR mix nn c cho vo mt PCR mix cho mi ln
chy PCR v cc mc ch sau:

Kim tra nhy ca PCR mix ang s dng xem c t khng nu chng [+]
c cho vo PCR mix vi nng LOD cho c sn phm khuch i pht
hin c. PCR mix c cho l t nhy khi chng [+] cho kt qu [+] pht
hin c bng cc phng php trn, v khng t nhy khi chng [+] cho kt
qu [-] tnh.

i vi PCR pht hin sn phm khuch i bng in di th kt qu in di

sn phm PCR ca chng [+] s cho vch in di ng kch thc mong i, nh
vy ngi c kt qu c th d dng so snh vi cc vch in di sn phm PCR
ca mu bnh phm kt lun mu bnh phm c kt qu dng tnh (khi cho
vch in di trng vi kch thc vch in di ca sn phm PCR ca chng [+])
hay m tnh (khi cho vch in di khng trng vi kch thc vch in di ca sn
phm PCR ca chng [+] hay khng c vch in di).

Tuy nhin chng [+] cho PCR mix khng kim tra c qui trnh tch chit
nucleic acid t mu c t hay khng. Mun kim tra c vn ny th ngi
thc hin th nghim phi c mt mu bnh phm bit trc [+] hay mt mu
gi bnh phm [+] thc hin tch chit nucleic acid mu ny song song vi cc
mu bnh phm tht.

V cc mc ch nh vy nn khi thc hin th nghim PCR, ngi thc hin th

nghim nn lun dnh mt PCR mix cho chng [+].

130
 Chng [-] cho PCR mix

nh ngha: Chng [-] cho PCR mix l mu nc tinh sch hay TE 1X c cho
vo PCR mix trong mi ln lm xt nghim PCR/real-time PCR chn on.

Vai tr: Chng [-] cho PCR mix nn c cho vo mt PCR mix cho mi ln chy
PCR, v cho vo cui cng sau khi cho mu v chng [+] vo cc PCR mix tng
ng v cc mc ch sau:

Chng minh thao tc cho mu v chng [+] vo cc PCR mix khng b nhim
cho hay khng b nhim sn phm khuch i v nu chng [-] cho PCR mix li
c kt qu [+] sn phm khuch i th c th cc thao tc trn b nhim cho hay
ngoi nhim sn phm khuch i.

Chng minh PCR mix ang s dng l hon ton tinh sch khng b nhim cc
DNA ngoi lai v nu chng [-] li c kt qu sn phm khuch i khng c
hiu [+] th chng t PCR mix b nhim cc DNA ngoi lai.

Tuy nhin chng [-] cho PCR mix khng kim tra c ton b qui trnh thc
hin th nghim PCR c b ngoi nhim sn phm khuch i hay nhim cho
khng. Mun kim tra c ton b qui trnh thc hin th nghim PCR th ngi
thc hin th nghim phi thc hin qui trnh PCR trn mt mu th bit trc
[-] tnh v thc hin song song vi cc mu bnh phm.

V cc mc ch nh vy nn khi thc hin th nghim PCR, ngi thc hin th

nghim nn lun dnh mt PCR mix cho chng [-].

Chng ni ti (Internal control, IC)

nh ngha: L DNA c khuch i song song vi DNA ch nhng cho sn

phm khuch i c: (1) kch thc khc (trong PCR kinh in) sn phm khuch i
ca DNA ch nh vy c th phn bit c khi in di; v/hay (2) trnh t khc hon
ton hay khc mt phn (trong PCR-ELISA hay trong real-time PCR) nh vy c th
phn bit c khi s dng d c hiu (specific probe).

Cc loi chng ni ti: C 4 loi chng ni ti sau y, ty ngun gc v thit k:

Chng ni ti l DNA b gen vt ch (hnh 63-A), v d b globin gene nu

bnh phm l m ly t ngi, hay myoglobin gene ca tm nu bnh phm l cc
131
 mu tm s. c th khuch i c DNA chng ni ti, chng ta phi cho
thm vo trong PCR mix mt cp mi c hiu vi DNA chng ni ti v nn
thit k mi sao cho nhit bt cp tng t nhit bt cp ca mi dnh cho
DNA ch, v khng to ra cc dimer primer vi mi ca DNA ch. Do s dng
DNA b gene vt ch nn chng ni ti ny kim sot c mu th c khng,
kim sot c h thng tch chit nucleic acid trn mu th c tt khng, kim
sot c c ch c hay khng. Tuy nhin loi chng ni ti ny khng kim sot
c h thng khuch i DNA ch v dng mi khc bit v ch dng cho mt
s bnh phm c cha lng t bo m vt ch.

B
A

C D

Hnh 63: Cc lai chng ni ti (A) l b gene vt ch; (B) l DNA tng hp c ngun gc t DNA
ch; (c) DNA tng hp c ngun khc DNA ch nhng s dng cng mi DNA ch; (D)
DNA khc bit han tan vi DNA ch

Chng ni ti l DNA tng hp c ngun gc l DNA ch (hnh 63-B), l

DNA c tng hp bng cch dng ng sn phm khuch i ca DNA ch
nhng ct ngn i hay chn mt on DNA nh lm cho n di hn. Chng ni
ti loi ny s dng cng mi vi DNA ch nn khng cn thit k mi cho
thm vo PCR mix.

Chng ni ti l DNA tng hp c ngun gc khc DNA ch nhng s dng

cng mi vi DNA ch (hnh 63-C), l DNA c tng hp bng cch dng
132
 sn phm khuch i ca DNA khc bit vi DNA ch nhng chn hai u cc
trnh t ging ht trnh t mi ca DNA ch. Do s dng cng mi vi DNA ch
nn khng cn thit k mi cho thm vo PCR mix.

Chng ni ti l mt h thng mi v DNA khc bit vi DNA ch (hnh 63-

D), c khuch i song song vi DNA ch nhng dng mi khc bit DNA
ch v DNA chng ni ti c ngun gc khc bit hon ton vi DNA ch.
c th khuch i c DNA chng ni ti, phi cho thm vo trong PCR mix mt
cp mi c hiu vi DNA chng ni ti v nn thit k mi sao cho nhit bt
cp tng t nhit bt cp ca mi dnh cho DNA ch, v khng to ra cc
dimer primer vi mi ca DNA ch.

Vai tr chng ni ti: Tu c cho vo cc giai on no m chng ni ti c th

c dng cho cc vai tr sau:

Kim sot c s tch chit trn mu bnh phm tt hay khng nu c cho
vo bnh phm lng ti thiu va (LOD) hay c sn trong bnh phm.

Kim sot c c ch xc nh c kt qu l m tnh tht hay m tnh

gi do c c ch cn hin din trong cc mu tch chit nu c cho vo PCR
mix nng LOD trc hay sau khi cho tch chit DNA t bnh phm

Kim sot c h thng khuch i DNA ch c t c nhy hay

khng nu chng ni ti dng chung mi vi DNA ch v hin din trong mu
th hay cho vo trong PCR mix nng LOD.

Kim sot c h thng tch chit ang dng c tt hay khng v thao tc c
ngoi nhim khng nu c sn trong mu tht lm chng m hay c cho vo
mu tht lm chng m c tham gia tch chit cng vi mu bnh phm

h. Cc vn k thut cn bit khi c v phn tch mt kt qu real-time PCR nh

lng s dng mi v taqman probe

Sau khi han tt chng trnh chy real-time PCR trn my, phn tch c kt
qu nh lng chnh xc l mt vn rt quan trng i hi ngi phn tch phi
hiu r cc vn l thuyt cng nh k thut real-time PCR. y chng ti xin
trnh by cc bc phn tch mt kt qu real-time PCR vi mi c hiu v Taqman
probe gn mu FAM pht hynh quang dnh pht hin/nh lng DNA ch, cn
133
Taqman probe gn mu Hex pht hynh quang dnh pht hin sn phm khuch i t
chng ni ti s dng chung mi DNA ch. Phn tch kt qu nn theo cc bc sau

Trc ht chn ging chng [-]: Ch chn ging phn tch l ging chng [-].
Chn knh hynh quang Fam, chng ta s thy ging chng [-] c ng biu din
khuch i khng vt qua khi ng hynh quang nn (baseline) tc l m tnh.
Nu knh Fam m chng [-] c ng biu din khuch i vt qua khi baseline
(dng tnh) th c nguy c ngoi nhim trong qu trnh cho mu vo cc PCR mix. B
knh hynh quang Fam, chn knh hynh quang Hex, lc ny chng [-] c ng
biu din khuch i vt qua khi baseline (dng tnh). Nu knh Hex m
chng [-] cho ng biu din khuch i khng vt qua khi baseline (m tnh) th
cng c nguy c l PCR mix khng nhy cm cho khuch i/pht hin DNA chng
ni ti hay chng ni ti cho vo b h (khi trong tt c cc ging khc, ng biu
din khuch i knh Hex u m tnh, tnh hung ny phi yu cu nh sn xut
cung cp li chng ni ti).

nh ng chun: Ch
chn knh hynh quang
Fam l knh hynh quang
cho taqman probe ca DNA
ch, khng chn knh
Hex. Chn cc ging
chun v ging chng [-],
ng thi loi tr cc ging
c mu th cng nh cc
ging khc. Sau cho cc
gi tr s bn sao (copies) Biu 17: Mt biu chun c nh gi t khi c E trong khang
90-105%, cn R20.99. Phi c biu chun t mi tip
DNA ch chun ban u tc phn tch kt qu

vo cc ging cha cc pha long DNA chun. Sau khi cho cc gi tr s copies
vo cc ging chun, chng ta my chn ng baseline theo ch t ng c
biu chun (gi l standard graph). Nu ng baseline m my chn theo ch t
ng ct phi ng biu din ca chng [-] th ngi s dng trc ht chnh baseline
cycle (cc chu k nn) ca ging chng [-] xem ng biu din chng [-] c h

134
xung khng, nu khng c th c th phi nng ng baseline ln ng
baseline ny khng ct ng biu din ca chng m. nh gi biu chun l
tng nht (biu 17) l biu cho h s tng quan (R) l 0.990; ng thi hiu
qu PCR (PCR efficiency = E) t 90% n 105%, dc (slope) ca ng biu din
trong khang -3.4 n -3.6. Nu ng biu din khng t th chng ta, trc ht xem
li tng ging chun iu chnh baseline cycle ca tng ging kt hp vi vic nng
ln hay h xung ng baseline c th a cc gi tr R v E v cc tr s l tng.
Nu vn cha a c R v E v cc gi tr l tng th c th thay i gi tr ca cc
tr s copies trong khang cho php khng qu mt pha lang (v d nu gi tr ghi
l 10 copies, c th thay gi tr t 10 n 99; hay nu gi tr ghi l 100 th c th thay
gi tr t 100 n 999) chng ta c c ng biu din chun t l tng (tuy
nhin ch lm cch ny mt cch bt kh khng thi v tht ra nu phi thay i gi tr
s copies chun c ngha l chng ta phi chp nhn sai s khi thao tc pipetting ri,
m nu chp nhn nh vy th chng ta cng s phi chp nhn c sai s trong kt qu
nh lng). Chuyn biu khuch i sang dng log c nh c ng nn ri
sau chuyn li dng graph bnh thng. Ghi nhn tr s baseline ca ng chun.
Lu : Vic thc hin PCR cc mu chun c hai ngha: (1) Cho c ng chun
lm c s tnh ton s copies ban u ca mu th; (2) Kim sot c thao tc
pipetting ca ngi lm th nghim c tt khng, qua tr s R v E c t khng. Do
vy m ngi lm th nghim bt buc phi cho cc mu chun v chy PCR. Mt s
cng ty trong nc khng hiu c ngha ca vic ny nn h cung cp chun
cho sn vo PCRmix, v lm nh vy th kt qu nh lng chc chn s khng m
bo c s chnh xc v khng th kim sot c thao tc pipetting ca ngi lm
xt nghim c tt khng.

nh s lng copies ban u c trong cc ging cha mu th: Sau khi c

ng chun, xc nh s lng copies ban u (SQ = starting quantity) ca DNA ch
trong cc ging mu th bng cch chn tng ging vo phn tch cng vi cc ging
chun.

i vi cc mu c ng biu din khuch i ct c ng baseline th s

lng nh lng c chnh l SQ c trong th tch mu a vo phn ng (biu
18). tnh c s copies trong 1ml huyt thanh, chng ta s nhn SQ vi h s K (l

135
h s c lin quan n thu hi sau tch chit cc mu chun bit r s copy c chn
DNA).

Biu 18: Mu c ng biu din khuch i ngc ln ct c baseline l mu nh lng dng tnh. Tr s
nh lng SQ c xc nh l s copies c trong th tch mu th cho vo ng phn ng. S lng
copiesDNA c trong 1 ml mu bnh nhn c tnh bng cch nhn SQ vi 50

Biu 19: Mu ch c th kt lun m tnh (ng biu din khuch i c bng kng FAM khng ngc ln) hay
di ngng pht hin (ng biu din khuch i c bng kng FAM ngc ln nhng khng ct
c ng baseline) khi c tn hiu khuch i ca chng ni ti c c bng knh hynh quang
HEX

i vi cc mu m ng biu din khuch i khng ngc ln c hay ngc

ln c nhng vn khng ct c baseline th chn thm knh mu Hex xem c
ng biu din khuch i ca DNA chng ni ti hay khng? Nu c th kt lun l
mu m tnh i vi cc mu c ng biu din khuch i khng ngc ln c
(biu 19) v tr li l mu di ngng pht hin i vi cc mu c ng biu
din khuch i ngc ln c nhng khng ct c ng baseline. Nu khng c
ng biu din khuch i ca chng ni ti th c th c kh nng l mu cn cht
c ch, nn thc hin li tch chit, nu mu vn cn b c ch (khng c tn hiu
136
khuch i ca DNA chng ni ti) th kt lun l mu b c ch, xin mu khc
lm li. i vi cc mu c ng biu din khuch i ct ng baseline qu sm
hay l ngc ln qu sm ct ng baseline ri li khng ngc ln thm na, chn
ging ny, chnh li baseline cycle v c khi my t ng chn khang baseline cycle
qu ngn nn lm mu c tn hiu khuch i ngc ln qu sm. Nu sau khi chn
li cc mu ny c ng biu din khuch i ging cc mu m tnh th phn tch
nh cc mu m tnh. Nu c ng biu din khuch i ging cc mu dng tnh
th phn tch ging cc mu dng tnh.

i. Tr li kt qu PCR/real-time PCR chun mc

PCR/real-time PCR l mt xt nghim rt nhy cm v c hiu. Tuy nhin v cht

lng xt nghim rt tu thuc vo phng php v trnh thc hin xt nghim, do
vy u im ny ch t c khi ngi lm xt nghim c tri thc kim sot
c cc s st trong qu trnh thc hin xt nghim thng qua cc chun mc bt
buc phi c khi lm xt nghim.

Chng [+] phi cho kt qu [+]

chng minh khu khuch i t nhy

Chng [-] phi cho kt qu [-]vi ch nhng

phi cho kt qu [+] vi chng ni ti
chng minh khu tch chit v khuch i t nhy
v qu trnh lm xt nghim khng b ngoi nhim

Mu mun kt lun [-] phi cho kt qu [+] vi

chng ni ti v kt qu [-] vi ch
chng minh mu khng b c ch

Hnh 64: Mt kt qu PCR nh tnh chun mc phi th hin c tt c cc im kim sot qu trnh lm
xt nghim nh c gii thch

Ngoi ra mt i hi rt quan trong na i vi real-time PCR nh lng, l

phi c kt qu nh lng chnh xc. Trong y sinh hc, bt c mt xt nghim nh
lng no cng i hi phi chy mu th cng lc vi mu chun. Mu chun trong
PCR nh lng phi cho kt qu t tuyn tnh cao vi hiu qu PCR tuyt i
chng minh ngi lm xt nghim thao tc pipetting chun, khng c s st. Khng

137
ch phi bit s dng cc chun mc kim sot cht lng ca cc khu khi lm xt
nghim, m ngay c trong khu tr li kt qu xt nghim, ngi lm th nghim cng
phi th hin c cc kim sot ny trn phiu tr li.

Mt kt qu xt
nghim PCR nh tnh n Khuch i cc nng
chun ca trnh t ch
Khuch i chng ni
tay ngi ch nh xt ti c pht hin nh
taqman probe c hiu Khuch i trnh t
gip ngi s dng kim chng ni ti
nghim s khng c sat c c ch trong
mu c hay khng
Khng c
xem l chun mc nu trn khuch i ch

phiu tr li kt qu khng
th hin v chng minh
c cc kim sot c
Cc chun gip kim sat chnh xc pipetting khi
thc hin trong qu trnh biu chun t R0.99 and E=90-105% v gip nh
lng chnh xc s copies ch c trong cc mu th

lm xt nghim. Hnh 64
minh ha v ct ngha cc
chun mc phi th hin
trn kt qu xt nghim
PCR nh tnh. Biu 20: Minh ha mt trnh by kt qu PCR nh lng chun mc
vi biu chun chng minh mu lun chy chung vi
Mt kt qu xt chun v thao tc pipetting c kim sot chun mc (B),
nh vy m kt qu nh lng l chnh xc (A)
nghim PCR nh lng
khi n tay ngi ch nh xt nghim s khng c xem l chun mc nu trn phiu
tr li kt qu khng th hin v chng minh c l

(i) mu th c chy chung vi mu chun qua ng biu din chun c v tr

ca mu th,

(ii) thao tc nh lng chun mc qua hiu qu PCR v h s tng quan trn
biu chun t cc thng s l tng, v

(iii) nu mu kt lun m tnh hay di ngng phi c dng biu din khuch
i ca chng ni ti. Cc biu 20-A v 20-B minh ha v ct ngha cc chun mc
phi th hin trn kt qu xt nghim real-time PCR nh lng

Thao tc th nghim chun mc v th hin kt qu chun mc, mi l

PCR/real-time PCR chn on.

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j. Cc vn k thut cn bit khi s dng mt kt qu PCR/real-time PCR chn
on

PCR/real-time PCR l mt cng c tuyt vi dng trong chn on v nhy

cm kh c th c mt xt nghim no snh kp, ng thi c hiu ca PCR/real-
time PCR trong chn on pht hin cc tc nhn vi sinh gy bnh cng khng khc g
phng php vi sinh nui cy. Tuy nhin vic s dng kt qu PCR/real-time PCR c
hiu qu hay khng l rt ty thuc vo s hiu bit ca ngi s dng kt qu v cho
ch nh lm xt nghim. Chng ti xin a ra mt s minh ha trong s dng kt qu
PCR/real-time PCR pht hin tc nhn vi sinh gy bnh cho ngi

Kt qu PCR/real-time PCR [+] c xc nh c ngi b th nhim tc nhn vi

sinh gy bnh?: ng nh vy v PCR/real-time PCR pht hin trc tip tc nhn vi
sinh gy bnh thng qua pht hin cc nucleic acid ch c hiu vi sinh vt c mt
trong bnh phm ly t bnh nhn. Tuy xc nh c l ngi b th th nhim tc
nhn vi sinh gy bnh nhng c xc nh c ngi b bnh hay khng c
iu tr l mt vn khc. Ly v d mt ngi th m pht hin vi khun lao bng
th nghim PCR, kt qu [+], bc s vn cha nn cho ch nh iu tr lao m phi
xem th X quang phn c thm nhim r nh phi tiu biu cho nhim lao hay
khng ri mi iu tr. Nu X quang phi vn trong v bnh thng th ch nn theo di
tip quan xt nghim PCR v qua X quang phi xem c din tin n lao hay khng
tong vng 1-3 thng, nu khng din tin n lao th khng cn thit iu tr cho
bnh nhn.

Kt qu PCR/real-time PCR [-] c xc nh c ngi b th khng mang tc

nhn gy bnh khng? Khng chc nh vy v PCR/real-time PCR khng th cho gi
tr tin on m (negative predictive value, NPV) t 100% cho d ly mu chnh xc
v th nghim chnh xc, v kt qu cn ty thuc rt nhiu vo din tin ca tc nhn
gy bnh trong c th ngi th. V d mt ngi HbsAg [+], th HBV-DNA cho kt
qu [-], c th ni ngi khng nhim HBV hay khng? Chc chn l khng, v
ngi vn cn mang HBV trong gan v ang trong giai an m min dch khng
c hiu ca c th cn t ch c virus nn HBV ch trong gan v to ra cc virus
khng han chnh l cc khng nguyn b mt khng c li DNA m thi. y chnh
l cc trng hp ngi lnh mang HBV, vn l ngi ang b nhim HBV v vn c

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 nguy c ly nhim cho ngi khc. Do vy chng ta cng rt thng gp nhiu kt qu
HBV-DNA [-] trn bnh nhn HBsAg [+].

S dng kt qu real-time PCR nh lng nh th no cho ng? y cng l

mt vn cn bn. Chng ta u bit real-time PCR dng trong nh lng tc nhn
vi sinh ch, nay gi l qPCR, rt c gi tr nh gi v theo di c hiu qu iu
tr cc bnh nhim virus nh HBV, HCV, HIV. Tuy nhin lm th no bit c s
khc bit v kt qu nh lng l c ngha? C nhiu ngi nhm ln vn ny
nn cho nh gi hay nhn xt sai lm v hiu qu iu tr. Kt qu nh lng ch
c coi l khc bit khi s khc bit gia hai ln th l trn hay bng 100 ln, gi l 2
log10. L do phi 2 log10 l v khi nh lng ngi ta xy dng ng chun da
vo cc pha long theo h s 10 ca cc nng DNA chun, v do sai s c th
chp nhn c l trong vng 1 pha lang nn kt qu ch c xem l khc bit khi
cch nhau 2 pha lang, tc l 2 log10. Chnh v vy, trnh cho cc nh lm sng
nh gi sai, hin nay trn th gii cc nh bnh hc chp nhn tr li kt qu l log10
ch khng phi l s lng copy chnh xc. V d kt qu l 425,379,000 copy th nay
s tr li l 4.25 log 8, hay n gin l log 8. Nh vy nu kt qu ln 2 l 117,318,000
copy th s tr li l 1.17 log 8, hay n gin l log 8. Vi cch tr li ny bc s iu
tr s thy c l lng virus cha gim sau thi gian iu tr. Ngoi ra, nh iu tr
mt khi hiu c vn ny, h s khng ch nh cho bnh nhn lm xt nghim
nh lng trong thi gian qu ngn ch trong vi tun sau khi xt nghim nh lng,
v chc chn ch trong thi gian ngn nh vy rt kh nh gi v so snh kt qu
nh lng vi nhau.

Xy dng mt phng th nghim PCR/real-time PCR chn on

Mt phng th nghim PCR chn on s bao gm cc phng v khu vc lm vic nh

sau:

1. Phng tch chit nucleic acid

y l phng lm nhim v tch chit nucleic acid v cho cc mu tch chit vo

cc PCR/real-time PCR mix chun b cho vo my chy PCR.

Phng ny cn trang b cc thit b v dng c sau:

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 Mt bung thao tc PCR l mt t thao tc kh tnh c trang b n chiu sng v
n cc tm, bung ny c th t t cc hng sn xut nc ngoi hay t ng (hnh
65). Kch thc ti thiu cho 2 ngi lm vic. Trong bung thao tc ny phi trang b
cc dng c v thit b nh sau: vortex x 2, my nhit kh x 2, my ht chn khng x
1, micropipette 1000ul x 2, micropipette 20-200ul x 2, micropipette 1-20ul x 1, l
vt liu d, nhim x 2.

Cc thit b khc phi c: my ly tm 13,000 x 2, my ly tm lnh 13,000 x 1, my

ly tm bn 4000 cho tube 15ml x 1, t ng -18 n -30oC vi s lng ty vo mu
nhiu hay t, t mt # 280 lt cng vi s lng ty mu nhiu hay t.

2. Phng t my PCR v real-time PCR

y l phng lm nhim v chy my PCR v real-time PCR. Phng ny cn

trang b my real-time PCR v my PCR thng vi s lng ty mu nhiu hay t.
lm chn on th nn t my ca cc hng c giy php quc t (lisence) c cung
cp my PCR/real-time PCR dng cho chn on v hin nay Biorad l cng ty c
c giy php ny.

m bo my chy n nh, khng b tt khi cp in gia chng th nn trang

b b lu in 6KVA v phi c dy ni t.

Phng t my PCR nn thng vi phng tch chit nucleic acid.

3. Phng in di c kt qu

y l phng lm nhim v ch cc gel in di, thc hin in di v c kt qu

in di. Phng ny phi trang b cc thit b v dng c sau:

Mt bung thao tc in di: l mt t thao tc kh tnh c trang b n chiu sng

v n cc tm, bung ny c th t t cc hng sn xut nc ngoi hay t ng
(hnh 65). Kch thc ti thiu cho 1 ngi lm vic. Trong bung thao tc ny phi
trang b cc dng c v thit b nh sau: vortex x 1, b in di agarose loi minigel x 2,
my ly tm compact x 1, micropipette 1-20ul x 1, hp nha ng gel dng xong x 1,
hp nha ng gel cha/ang dng x 1, hp nha ng u pipette d x 1.

Cc dng c v thit b khc phi c l: mt l vi sng, mt h thng chp nh gel,

mt t gel, mt cn in t cn agarose.

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 Phng in di c kt qu nn tch bit hay ch thng c vi phng t my
PCR/real-time PCR

4. Phng pha ch thuc th PCR v real-time PCR

Ch c cc phng th nghim chn on ln mi nn c phng ny, v phi tch

rt ra 3 phng ni trn. Phng ny trc khi vo phi thay o bng o chuyn ch dng
trong phng ny m thi. Cc thit b, ha cht, dng c pha ch trong phng,
khng c em ra ngoi. Ni chung y l phng phi tinh sch. Cc thit b v dng
c trong phng nn c l:

T thao tc pha ch thuc th, nn l cc clean bench dnh cho 2 ngi c trang b
n cc tm v n chiu sng. Trong t ny trang b: vortex x 1, my nhit kh x 1,
my ly tm 13000 x 1, micropipette 1000ul x 2, micropipette 20-200ul x 2,
micropipette 1-20ul x 1, l vt liu d, nhim x 2.

T thao tc cn chnh pH, cng nn l cc clean bench dnh cho 1 hay 2 ngi c
trang b n cc tm v n chiu sng, bn trong c: cn in t x 1, my o pH x1,
khuy t x 1, micropipette 1000ul x 1, micropipette 200ul x 1, micropipette 20ul x1.

T thao tc pha cc ha cht c, nn l t an tan sinh hc lp 1 bn trong cng

trang b cn in t x 1, my o pH x1, khuy t x 1, micropipette 1000ul x 1,
micropipette 200ul x 1, micropipette 20ul x 1, pipette aid x 1.

Cc thit b khc, c s lng ty qui m, nh my lm vy, my ly tm 13,000

x1, my ly tm bn 4000 cho tube 15ml x 1, t ng -18 n -30oC vi s lng ty
vo mu nhiu hay t, t mt # 280 lt cng vi s lng ty mu nhiu hay t.

Di y minh ha mt phng th nghim sinh hc phn t chn on ln bao gm c

PCR/real-time PCR v gii trnh t (hnh 66, 67) dnh cho mt bnh vin ln ca thnh
ph H Ch Minh m chng ti gi thit k. Ngoi ra chng ti cng a ra thit k
phng th nghim PCR/real-time PCR cho cc phng th nghim trung bnh (hnh 68)

142
 Hnh 65: M hnh mt t thao tc PCR t ng

Hnh 66: S phng pha ch cc thuc th sinh hc phn t

143
 Hnh 67: S phng tch chit nucleic acid v phng in di kt
hp phng gii trnh t

144
Hnh 68: S mt phng th nghim PCR v real-time PCR dnh cho phng th nghim lm
sng ti cc bnh vin tuyn thnh ph hay tnh

145