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PCR Vandekythuat
PCR Vandekythuat
PCR Vandekythuat
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.
116
2. Cc vn k thut cn quan tm
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.
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.
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).
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.
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.
121
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).
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.
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...
123
e. Cc vn k thut trong th nghim in di trn thch c kt qu
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.
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.
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.
125
f. Bin php an tan khi s dng ethidium bromide
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.
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.
129
g. Vai tr ca chng [+], chng [-] v chng ni ti
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.
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.
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.
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
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 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
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.
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
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
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
(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
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j. Cc vn k thut cn bit khi s dng mt kt qu PCR/real-time PCR chn
on
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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 [+].
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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.
3. Phng in di c kt qu
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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
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.
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Hnh 65: M hnh mt t thao tc PCR t ng
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Hnh 67: S phng tch chit nucleic acid v phng in di kt
hp phng gii trnh t
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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
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