PPDH LAB .

DIAGNOSTIK
FKH IPB

RT-PCR
(REALTIME POLYMERASE CHAIN
REACTION)
Disusun oleh:
Yu n i t a A m a n d a M ( B 0 9 0 1 2 0 1 0 4 6 )
Avrita Reza M el y a na ( B0901201070 )
R T- P C R (Real T i m e Po l y m e ra s e C h a i n
R e a c ti o n ) a d a l a h a m p l i fi k a s i D N A
s e c a r a in vitro. Te k n i k P C R d i g u n a k a n
u n t u k m e n g a m p l i fi k a s i s e k u e n D N A
d a l a m jumlah ribuan atau jutaan kopi
dengan meihat peningkatan pada
fl u o r e s e n s i g n a l .

PPDH LAB . DIAGNOSTIK

FKH IPB
 P E N E N T U A N J U ML A H V IR U S

TUJUAN RT- D E TE K S I A D A N Y A MUTA S I GE N

PCR A N A L IS IS E K S P R E S I G E N

PPDH LAB . DIAGNOSTIK

FKH IPB
 K O M PO N EN
RT-
PCR
1.Te m p l a t e
2.Taq D N A Polymerase
3 . Reverse
Transcriptase
4 . Primers
5.Pro be
6.d N t Ps
7 . Buffer ( M g + ) PPDH LAB . DIAGNOSTIK
FKH IPB
INSTRUMEN

PPDH LAB . DIAGNOSTIK

FKH IPB
 5 0 µ L s a m p e l + 1 0 0 µ L b u ff e r lisis (plat e k s t ra k s i ) + 2 0 µ L l a r u ta n

Ekstraksi b e a d s m a g n e ti c
P l a t d i g e t a r k a n ( 4 m i n ) d a n d i d i a m k a n d i m a g n e ti c s t a n d ( 2 m i n )

RNA
untuk mengendapkan beads
Supernatant dibu an g d e n ga n m e n g g u n a k a n mikropipet
P l a t d i t u r u n k a n d ar i m a g n e ti c s t a n d + 1 0 0 µ L b u ff e r p e n c u c i 1 d a n

Virus d i g e t a r k a n (30s) s e t e l a h itu d i d i a m k a n d i a t a s m a g n e ti c s t a n d

(1 m i n ) lalu s u p e r n a t a n t d i b u a n g
P e n c u c i a n d i l a k u k a n d e n g a n b u ff e r p e n c u c i 2 s e b a n y a k 2 x
B e a d s d i k e r i n g k a n d e n g a n d i ge t a r k a n /a g i t a s i (2m in )
d i t a m b a h k a n b u ff e r e l u s i 5 0 µ L u n t u k m e l a r u t k a n R N A
P l a t e d i a g i t a s i ( 3 m in ) d a n d i d i a m k a n d i m a g n e ti c s t a n d (1
min)
untuk mengendapkan beads
S u p e r n a t a n R N A ( t e m p l a t e n R N A ) d i p i n d a h k a n d i p l ate b a r u d a n
d i s i m p a n k e d a l a m s t o r a g e - 7 0 d e ra j at

PPDH LAB . DIAGNOSTIK

FKH IPB
RT- R e a g e n P C R d i c a m p u r k a n k e d a l a m e p e n d o r ff d a n d i s i m p a n p a d a s t o r a g e - 2 0 C R N A s e -
f ree d H 2 O 1,08 μl, b u ff e r 2 X 12,50 μl, p r i m e r f o r w a r d (20μM) 0,25 μl, p r i m e r R e v e r s e
(20μM)0,25 μl, e n z i m m i x 2 5 X 1,00 μl, p ro b e ( 6 μ M ) 0,25 μl, d a n d e t e c ti o n e n h a n c e r 1,67 μl

PCR S e k u e n s primer/ p r o b e y a n g d i g u n a k a n f o r w a r d (M+25): 5 ’ - AG A T G A G T C T T C TA A C C G

A G G TCG-3’; m a t r i k s r e v e r s e (M-124): 5 ’ - TG C A A A A A C AT C T T C A A G T C T CTG-3’; p r o b e
A I V Matrix (M+64): 5’d FA M - T C A G G C C C C C T C A A A G C C GA-BHQ1-3’; H 5 F o r w a r d ( IVA
D148H5): 5 ’ - A A A C A G A G A G G A A AT A A G T G G A G T A A A ATT-3’; H 5 r e v e r s e (IVA-D149H5):
5 ’ - A A A G AT A G A C C A G C T A C C AT G AT T GC - 3 ’; p rob e I VA- H5a : 5’d FA M - T C A A C A G T G G C G
A G T T C C C TA G C A BHQ1-3’
K e d a l a m p l at o p ti c d i t a m b a h k a n 17 μl c a m p u r a n P C R + 8 μl t a m p l a t e R N A
P l a t o p ti c d i t u t u p d e n g a n s e a l o p ti c d a n d i m a s u k a n k e d a l a m m e s i n A p p l i e d B i o s y s t e m s
7 5 0 0 R T- P C R
M e s i n d i at u r d e n g a n s i k l u s : Ta h a p 1 (1×): R e v e r s e t ra n s k r i p s i 4 5 o C 1 0 m e n i t ; d e n a t u ra s i
9 5 o C 10 m e n i t . Ta h a p 2 (45×): D e n a t u r a s i 9 4 o C 1 deti k; a n n e a l i n g + e k s t e n s i 6 0 o C 3 0
d e ti k
H a s i l P C R d i a n a l i s i s m e n g g u n a k a n a p p l i e d b i o s y s t e m s 7 5 0 0 R T- P C R
s y s t e m s o ft w a r e (hasil l a y a k a p a b i l a nilai C t K + isolasi R N A ≤ 35, K + P C R ≤
25, d a n K - ti d a k m e m i l i k i nilai C t

PPDH LAB . DIAGNOSTIK

FKH IPB
T h e r m a l Cycle

PPDH LAB . DIAGNOSTIK

FKH IPB