Vai tr v ngha cc xt nghim

sinh hc phn t trong trong nghin

cu v chn on bnh nhim trng
Phm Hng Vn*

*Bc S Y Khoa, Tin S Y Hc. Ging vin B Mn Vi Sinh, Khoa Y, i Hc Y Dc TP. HCM,
Ph Phng Th Nghim Trung Tm i Hc Y Dc TP.HCM.

Cn xt nghim kp thi v nhy cm

hn chn on pht hin
cc tc nhn vi sinh gy bnh

Xt nghim PCR cho Lao

q
q

Xt nghim tm AFB khng

xc nh c lao
Kh nhiu trng hp lao
phi m soi khng thy, cy
khng ra
Cc trng hp lao ngoi
phi rt kh chn on xc
nh nh lao mng no, lao
khp, lao mng bng, lao
mng tim, lao xng
Phi c kt qu khng sinh
sm bit c a khng
khng

TB-PCR c nghin cu
nh gi l hu dng lm sng
Pulmonary TB
Se
Sp
Extrapul. TB
Se
Sp
TB meningitis
Se
Sp

PCR

Culture

AF staining

94%
97%

77%
99%

64%
90%

75%
99%

67%
100%

27%
61%

48%
100%

39%
100%

9%
100%

KIT, Amsterdam, 1996

Pht hin BK
So snh PCR vi ZN trn 132 mu m
PCR
Dng
22

m
1

23
(17%)

17

92

109

39

93

132

(30%)

Nguyn Vn Thnh - 2002

RT-PCR chn on sm
st Dengue v SXH Dengue

 nhy ca MAC-ELISA theo ngy bnh

100
90
80
70
60
50
40
30
20
10
0

100%

72%
56%
39%

N4

N5

N6

N11
Vu Th Que Hng (2000)

Viremia at rat cao trong

nhng ngay au

IgG (cac lan sau)

(Keo dai suot i)

2 3

6 7

IgG (lan au)

(Keo dai suot i)

8 9 10 11 12 13 14 15 16 17 18
IgM
(Keo dai 30-60 ngay)

-3 -2 -1 0 1

2 3

6 7

8 9 10 11 12 13 14 15 16 17 18

100 benh nhan SXH

So sanh ket qua gia PCR va IgM theo ngay benh

PCR

IgM

100
80

99%

95%

60
40

75%

85%

84%

61%

20
0

PCR co kha nang phat hien SXH ngay trong nhng

ngay au cua benh
N2 (n=31)

N3 (n=32)

N4-N7 (n=37)
Nguyen Van Thnh va CS., 2004

100 benh nhan SXH

So sanh ket qua gia PCR va IgM theo tieu cau

PCR

100
90
80
70
60
50
40
30
20
10
0

IgM

93%
98%

88%

82%
67%

38%
PCR co kha nang phat hien SXH ngay ca khi cha co
bien oi so lng TCau
<100.000/mm3

100.000150.000/mm3

>150.000/mm3
Nguyen Van Thnh va CS., 2004

Cn mt gii php ton din

chn on/theo di iu tr
vim gan B v vim gan C mn tnh

Nhim vim gan siu vi B (HBV)

WHO 2001

Xt nghim sinh hc phn t cho vim gan B

HBsAg [+]
HBVDNA [+]
Pht hin t bin precore

PCR pht hin HBV-DNA

v nh lng

qHBVDNA
105
ALT

Ann Intern Med. 2002;137:1-9. Updated Definitions of Healthy

Ranges for Serum Alanine Aminotransferase Levels. Daniele
Prati, MD; Emanuela Taioli, MD, PhD; et Al.

ALT 19u/ml: N
ALT 33u/ml: Nam

ALT

ALT Normal

iu tr

Biopsie
or Fibroscan
AFP

qHBVDNA/3th
105

qPCR nh lng
HBV-DNA

LamR, AdfR

Pht hin t bin precore

Bt thng
Pht hin t bin khng thuc

Nhim vim gan siu vi C (HCV)

Schering-Plough

Din tin nhim HCV

Nhim HCV (100)
(Phase cp)

85% (85)
Mn tnh
15% (15)
Khi

20% (17)
X gan

80% (68)
n nh
75% (13)
Din tin chm

25% (4.5)
Ung th gan
X gan tin trin

Xt nghim sinh hc phn t cho vim gan C

nh lng
HCV genotype

RT-qPCR nh lng HCV-RNA

Tun 12 (4)
nh lng

RT-qPCR nh lng HCV-RNA

nh genotype HCV

Gim hn 2 log10 hay m tnh

HCV-RNA

Khng gim hn 2 log10

Cn nhc ngng
iu tr

Genotype - 1
iu tr 48 tun

Genotype non-1
iu tr 24 tun

(2002 NIH Consensus Conference)

RT-PCR phat hien HCV-RNA

Cui iu tr
HCV-RNA
Theo di sau 6 thng
HCV-RNA

HCV RNA (+)

Khng p ng

HCV RNA (+)

Ti pht

HCV RNA (-)

p ng

Thc t i hi cc nh khoa
hc, c bit l cc nh y hc
phi nhanh chng pht trin v
ng dng cc k thut sinh hc
phn t hin i, nht l PCR v
real-time PCR vo chn on v
h tr cho theo di iu tr bnh
nhn ti Vit Nam

PCR da trn nguyn tc khuch i ri pht hin an

DNA c hiu nn t c nhy v c hiu cao
Target DNA
4

1 DenaturaN (94oC)

2 Anealing (55-65oC)
3

3 Extension (72o C)

2n

PCR v real-time PCR l

mt k thut m nn v
ang tr thnh mt cng c
tt nht p dng c trong
chn an vi gi thnh
chp nhn c

PCR chnh la nuoi cay phan t khuech ai oan gen

Nuoi cay

PCR

Phong th nghiem PCR trong phat hien

tac nhan VSV gay benh chnh la phong
th nghiem vi sinh vi vai tro nuoi cay
phan t tng t phong th nghiem vi
sinh vi vai tro nuoi cay bo gene.
Do vay phat trien PCR phat hien tac
nhan vi sinh vat gay benh phai phat
trien tai phong th nghiem vi sinh

nh danh da vao kieu hnh sinh vat hoa hoc

bieu hien t cac kieu gen

Xac nh da vao kch thc oan gen

hay mot trnh t ac hieu cua oan gen

Pht hin v hay nh lng tc nhn vi sinh

vt gy nhim trng trn ngi v ng vt
q

Tc nhn virus cn phi pht hin sm c bin

php theo di (Dengue, HPV), iu tr (HSV), theo di
hiu qu iu tr (HIV/AIDS), hay phng nga (H5N1,
H1N1, SARS)
Tc nhn vi khun nhng cc phng php truyn
thng khng nhy, khng kp thi (Chlamydia, Lu
kinh nin)
Cc tc nhn gy dch v s khng an ton nu lm
xt nghim vi cc phng php vi sinh truyn thng
ti cc phng xt nghim lm sng cha c an ton
cp 3 (H5N1, H1N1, SARS, tc nhn khng b
sinh hc)

Lm th no p dng PCR
mt cch chnh xc v hiu qu?
PCR v real-time PCR l mt h thng m do vy
cho php ngi s dng tip cn bng cch thit k v
ch to cc thuc th hay b xt nghim dng trong
chn an. Tuy nhin v phi p dng cho chn an
cho nn khi tin hnh thc hin xt nghim PCR, ngi
lm xt nghim phi ch cc chun mc kim sat
cc sai lm trong qu trnh lm xt nghim v ngi
cho ch nh cng nh s dng kt qu cng phi quan
tm n cc cht lng ny cng nh quan tm n
cc nh gi ngha ca xt nghim

m bo cht lng khi

p dng PCR cho chn an

m bo cht lng ca vic

p dng PCR trong chn on
Ch nh xt nghim PCR
Ly v chuyn ch mu th n phng xt nghim

Thc hin xt nghim PCR ti phng th nghim

Phc trnh kt qu xt nghim n lm sng

Phn tch cc kt qu xt nghim

S dng cc kt qu trong chn on v iu tr

Qui trnh xt nghim PCR v cc im kim sat cht lng

Cc vn k thut trong
ly v gi mu th

Chun b mu th v tch chit

nucleic acid t mu th

Cc vn k thut trong
tch chit nucleic acid (NA)

Thc hin phn ng khuch i

nucleic acid (PCR/real-time PCR)

Cc vn k thut trong
khuch i NA

Lm th nghim c kt qu
Phn tch kt qu

Cc vn k thut trong th nghim

c KQ v trong phn tch KQ

S dng kt qu xt nghim
PCR/real-time PCR

Cc vn k thut trong
s dng kt qu

Vn ngn nga nhim cho v loi tr

ngoi nhim sn phm khuch i

Ly v chuyn mu th i lm
xt nghim PCR/realtime PCR

 m bo c mt xt nghim PCR
chnh xc th trc ht phi kim sat cho
c khu ly mu, vn chuyn mu, v
lu gi mu cho n khi lm xt nghim.

Ly, lu tr v chuyn ch mu th
ng v tr
q ng thi im
q ng cch
q ng vt cha
q

m bo c mt
VSV trong mu th
Trnh c ch
Tinh sch sinh hc,
dng mt ln

ng lu tr v chuyn ch
Lnh hay ng lnh

m bo c mt nucleic acid ca tc nhn vi sinh vt cn

pht hin v nucleic acid ny khng b ly gii hay b c ch

Vt liu ly mu

Cc kim sot cht lng ti phng xt nghim

Bnh phm

PCR

Kt qu

Tt c cc thuc th c pha ch hay cung cp

c kim tra cht lng phi c bng chng
kim tra cht lng cho thy phng php kim
tra nh th no, cch nh gi th no l t, v
kt qu kim tra cht lng ca thuc th .
Phi c cc chng dng bn vng gip
ngi s dng kim sot nhy ca cc thnh
phn trong b th nghim v qui trnh th nghim
Ngi s dng cc thuc th ny phi kim tra
khi nhn thuc th v lu li cho n khi dng
xong

Mt v d kt qu kim tra cht lng PCR mix dnh cho

PCR pht hin HBV v PCR pht hin M. tuberculosis

i vi ngi lm xt nghim th cng phi c cc

chng v chun kim tra cht lng ca cc
khu lm xt nghim bao gm c kim tra cht
lng ca cc thuc th ang dng c cn gi
c cht lng hay khng, kim tra cc bc xt
nghim c c st khng, v trong real-time PCR
cn phi nh gi chnh xc ca nh lng

Bnh phm
Kim sat nguy
c ngai nhim
trong qu trnh
tch chit NA

Chun b
Tch chit nucleic acid
Chy PCR

Kim sat nguy

c ngai nhim v
c ch trong qu
trnh khuch i

Kim sat
nhy ca khu
tch chit NA

Pht hin sn
phm PCR
Kim sat chnh xc
ca nh lng (qPCR

Kim sat
nhy ca qu
trnh khuch i

Internal Control
(IC)

STD-3

STD-2

STD-1

Internal Control
(IC)

NA extraction

PCR

C[-] + IC

STD-3

STD-2

STD-1

NA extraction

Sample +IC

Mu

C[-]

Mt xt nghim PCR nh tnh chun mc

Mt xt nghim qPCR nh lng chun mc

Gi tr
copies
ca
ng
chun

Mt xt nghim qPCR nh lng chun mc

Nu lm xt nghim PCR vi
Cc thuc th c kim tra cht lng vi
cc bng chng c lu gi, v
Khi lm xt nghim PCR c thc hin c
cc chng kim sat cht lng v kt qu
PCR cho thy cc chun kim sat cht lng
u vt qua c
Th kt qu PCR lun m bo c s chnh xc
t lc bnh phm c a vo xt nghim.

S dng kt qu PCR
Kt qu PCR [+] c cho l bn ang b bnh khng?

Xc nh c bnh nhn b nhim tc nhn gy bnh

Khng khng nh bnh nhn b bnh m cn ty thuc
nhiu yu t:
Vd: Bnh lao cn thm X-quang, lm sng
Vin gan B mn tnh cn thm s lng HBV-DNA
v men gan

S dng kt qu PCR
Kt qu PCR [-] c cho l bn khng b nhim?

Xc nh c tc nhn gy bnh khng c mt trong

bnh phm th nghim

Khng khng nh bnh nhn khng nhim tc nhn

gy bnh:
Vd: HBV-DNA [-] m HbsAg [+]
HCV-RNA [-] m anti-HCV [+]
HIV-RNA [-] m anti HIV [+]

S dng kt qu PCR
Kt qu nh lng nn phn tch nh th no?

Khng so snh s copies m ch nn so snh gi tr Log

v ch khng nh khc bit nh lng c ngha khi kt
qu hai ln th chnh nhau 100 ln

Vd: 425,379,000 copies so vi

4.25 Log 8 copies so vi

Nhn nh nh vy s trnh lm dng xt nghim nh

lng

117,318,000 copies
1.17 Log 8 copies

Cha kha cho cht lng PCR

p dng trong chn on

Thc t cng i hi cc nh khoa

hc, c bit l cc nh y hc
ngai k thut PCR, phi nhanh
chng pht trin v ng dng cc
k thut sinh hc phn t hin i
khc

Gii trnh t bng

in di mao qun

CEQ-8000

ABI 3130XL

Gii trnh t l h thng m rt sn sng cho cc xt

nghim chn on
Xc nh genotype
Pht hin t bin khng thuc (virus, vi khun)
Pht hin bnh l di truyn tin sanh cho thai nhi
Pht hin ung th nhy hay khng thuc
Pht hin v nh danh trc tip tc nhn vi sinh c
trong mu bnh phm m khng cn qua nui cy

Xc nh Genotype HCV
Phng php real-time
Phng php InoLIPA
Phng php gii trnh t

Real-time PCR c mt s c s
ng dng ti VN tht s cha ng tin cy

L do: Thit k probe cn cho cc genotype khc nhau

Xac nh genotype HCV

so sanh giai trnh t va InoLIPA (234 mau)

5NC

HCV cDNA
HCV2

HCV1

Real-time TQ
PCR

HCV3

Ethanol precipitation

PCR product
# 240bp
PCR sequencing

Kt hp nh lng v nh genotype
HCV (bng gii trnh t)

nh genotype HCV hu dng lm sng

ch c phng php gii trnh t 5-NC

Phn b genotype HCV

Kt qu 2000 mu thc hin ti NK-BIOTEK 2009

Xc nh genotype HBV
Tm t bin khng thuc ca HBV
BS nn cho ch nh tm t bin khng thuc v genotype HBV
khi bnh nhn khng p ng iu tr lamuvidine

Pht hin t bin khng thuc ca HBV

Phng php PCR-RFLP
Phng php real-time
Phng php gii trnh t

nh genotype HBV v
pht hin t bin khng thuc
K thut gii trnh t vng rt gene (Nam Khoa)
HBV DNA
264F

827R

95oC/5min
o
PCR94oC/30sec
55 C/30sec 40
72oC/1min X
72oC/10min

567bp
827R

Ethanol precipitation

PCR product

# 550bp

PCR sequencing
DTCS (Becman Coulter)

CEQ 8000

Pht hin khng Lamivudine, adefovir

gii trnh tgene PreS

Va pht hin nhiu v tr t bin

(k c heterozygote)

Va cho kt qu genotype

nh genotype HBV v pht hin .bin YMDD lamivudine-R

kt qu trn 455 mu thc hin 17/4/2007-17/4/2008

54.28

60
C
31%

50
40

B
69%

29.89

30
20

8.79

6.59

10

0.45

0
204

204+207

204+180

204+180+207

Khong B

Promoter
1762, 1764

T l phn b cc kiu t bin precore/core promoter pht hin

c trong 134 mu xt nghim ti NK-Biotek nm 2008

Quy trnh xt nghim vi khun k kh

kt hp vi sinh kinh in v gii trnh t gen 16S rRNA

Vi sinh kinh in
Ly v chuyn ch bnh

Bnh phm

Ly v chuyn
ch bnh phm
nh danh v
khng sinh

Nui cy hiu kh v k kh

Chn vi khun k kh

phm.
Nui cy phn lp tc
nhn nhim trng k kh.
Khng sinh bng
phng php pha long
khng sinh trong mi
trng lng

Gii trnh t gen 16S

rRNA nh danh vi
khun k kh.

Tc nhn nhim trng k kh phn lp trn

59 bnh nhn vim xoang hm

Peptostreptococcus:
P. micros (1)

Veillonella

11% (n=2)

P. stomatis (1)

Prevotella buccae

11% (n=2)
17% (n=3)

39% (n=7)

22% (n=4)

Streptococcus:
S. aginosus (1)
S. constellatus (3)
S. gordonii (3)

Propionibacterium acnes

Kim tra cht lng Probiotic dng cho ngi

Lun vn Thc S CNSH 2008

Tng kt cc kt qu pht hin v nh danh nm bng k thut PCR v gii

trnh t 28s rDNA trong cc mu bi nm ly t m vim xoang trn bnh nhn
Lun vn Thc S TMH 2008

Mt v d trong NSCLC
(Non Small Cell Lung Cancer)

NSCLC
(Non Small Cell Lung Cancer)
Tin on hiu qu tr liu ca EGFR tyrosine kinase inhibitor
(Gefitinib, erlotinib)
1. t bin gene (codon 19, 20, 21): PCR v sequencing (t
DNA cho tng exon hay tu mRNA cho nhiu exon)
2. nh lng gene EGFR: FISH, real-time PCR
3. nh lng biu hin gene (Ha m min dch, real-time
PCR nh lng mRNA

Pht hin t bin gene

Mu sinh thit ti (pht hin t bin trn
mRNA v trn tng exon ca gene)
Mu sinh thit paraffin bloc (pht hin t
bin trn tng exon ca gene)

Pht hin t bin gene

Kt lun
Sinh hc phn t ngy cng c nhiu ng
dng thc tin trong chn an v iu tr
Cc phng php tip cn khng phi
qu kh v trang thit b cng khng phi
qu t tin
Vn chnh yu l: (1) phi c con ngi
c kh nng xy dng k thut v t chc
phng th nghim, v (2) cc nh lm sng
phi c y tri thc ng dng