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REAL TIME PCR INDIAGNOSIS OF HUMAN
PARASITESRozliana Hanim Binti Mat Hasan
Journal Clu !"#$!#!$%" JPEP
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(&at is)uantitati*+,R+al Tim+
PCR--
• The Polymerase Chain Reaction
(PCR) is a process for the
amplification of specific fragments of
DNA.
• Real-Time PCR a specialized
techni!e that allo"s a PCR reactionto #e $is!alized %in real time& as the
reaction progresses.
• As "e "ill see' Real-Time PCRallo"s !s to meas!re min!te
amo!nts of DNA se!ences in a
sample
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Ho0 1o+sit 0or2-
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To understand real-time
PCR, let’s imagine
ourselves in a PCR
reaction tube at cycle
number 30…
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Ima4inin45PCR
• hat*s in o!r t!#e' at cycle
n!m#er +,
• A so!p of n!cleotides'primers' template'
amplicons' enzyme' etc.
• ',,,',,, copies of the
amplicon right no".
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/f "e "ere to graph the amo!nt of
DNA in o!r t!#e' from the start
!ntil right no"' at cycle +,' the
graph "o!ld loo0 li0e this1
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Ima4ini
n4 5PCR
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• A clump of !A t"e si#e of ten billion
planets $on’t %uite fit in our PCR tube
anymore&
• Realistically, at t"e c"ain reaction
progresses, it gets e'ponentially
"arder to find primers, and nucleotides&
And t"e polymerase is $earing out&
• (o e'ponential gro$t" does not go on
forever)
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Ima4inin4 5PCR
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• *f $e plot t"e amount of !A in our tube
going for$ard from cycle 30, $e see t"at it
actually loo+s li+e t"is
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Ima4inin4 5PCR
Ho0 0+4+t
&+r+--
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• e describe t"e position of t"e lines $it" a
value t"at represents t"e cycle number
$"ere t"e trace crosses an arbitrary
t"res"old&
• T"is is called t"e .Ct /alue&
• Ct values are directly related to t"e starting
%uantity of !A&
Ct value
a 9
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Ima4inin45PCR
Ho0 0+
4+t&+r+--
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&anim&asan'!$%" %%
T"e real-time soft$are converts t"e
fluorescent signals in eac" $ell to
meaningful data&
1& (et up PCR protocol&
2& (et up plate layout&
3& Collect data&
& Analy#e data&
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• Pro4r+ss o: r+a9tion is monitor+1 ; a9am+ra 1urin4 +a9& PCR 9;9l+#
• Fluorescent marker which bindsto the DNA- Probe
• Num+r o: 4+n+ 9ouor+s9+nt
• )uanti?9ation is a9&i+*+1 ;m+asurin4 t&+
in >uor+s9+nt 1urin4 +@
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• A s
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&anim&asan'!$%" %/
• PCR t&+rmal
9;9l+r >uorim+t+r• 5PCR
9on?4ur+1 to1+t+9t ! to 31i+r+nt>uor+s9+nt
9olour• Allo0
5uanti?9ation
o: % tar4+t
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Proin4 Alt+rnati*+s9ommonl; a
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A1*anta4+s o: 5PCR
• am
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Disa1*anta4+s o: 5PCR
• E@
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A
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Plasmodium spp.• B++n a
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Babesia spp.
• r+altim+ PCR assa; usin4 FRET PCRma; +
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Entamoeba spp.
• Di+r+ntiation o: Entamoeba histolytica an1Entamoeba dispar ; &;ri1ization
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Microsporodia spp.• multi
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&eishmania spp.
• RET= SBR 4r++n &as ++n us+1 to 1+t+9t an11i+r+ntiat+ 9ultur+1 strains o: Ol1 (orl1&eishmania spp# &eishmania ma'or, &eishmaniadono"ani, &eishmania tropica, an1 &eishmaniain$antum
Ni9olas et al
!$$!• r+altim+ PCR as+1 on tr;
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(&at is t&+ +st-
• T&+ o
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THAN OU
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REFLESI
APA!A" #$%$AN"&D$P !A '
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created 'inn and man!ind only to*orship me. *ant no pro"ision $rom
them, nor do *ant them to $eed Me
+ + - +/
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