real time pcr in diagnosis of human parasites

8/20/2019 Real Time Pcr in Diagnosis of Human Parasites

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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

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4+t&+r+--


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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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• 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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real time pcr in diagnosis of human parasites

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