Topi

c:PCR-
ELI
SA:Anemi
nentandemer
gingt
echni
quef
orcl
i
nicaldi
agnosi
sandi
t'
s

f
utur
epr
ospect
s

By-Preeti Singh and Nidhi Yadav

i

Depar
tmentofBi
osci
ences,
Cour
se:
MSCMi
crobi
ology
,

I
ntegr
alUni
ver
sit
y,Lucknow,
Indi
a

Abst
ract
:

PCRi
sawel
l
-est
abl
i
shedmet
hodf
ort
hequi
ckandef
fi
cientampl
i
ficat
ionofpar
ti
cul
ar

DNAsequences.Thecombi
nat
ionofPCRandELI
SAwasbor
noutofaneedf
orequal
l
y

qui
ck,sensi
ti
ve,andobj
ect
ivemet
hodst
odet
ectPCRpr
oduct
s.Thepol
ymer
asechai
n

r
eact
ion-
enzy
me l
i
nked i
mmunosor
bentassay (
PCR-
ELI
SA)i
s a semi
-quant
it
ati
ve

t
echni
quet
hati
nvol
vesdet
ect
ionofPCRpr
oduct
simmunol
ogi
cal
l
y,oncebi
oti
nyl
ated

DNA hasbeen i
mmobi
l
ized on a mi
cropl
ate.Thi
sappr
oach i
ssubst
ant
ial
l
ymor
e

sensi
ti
vet
hant
radi
ti
onalPCRsi
ncei
tident
if
iesnucl
eicaci
drat
hert
hanpr
otei
n.Wi
th

r
ecentadv
ancement
sinPCR-
ELI
SA,i
tisant
ici
pat
edt
hati
twi
l
lbemor
egener
all
y

r
ecogni
zed f
ori
ts hi
ghl
y sensi
ti
ve and r
api
d det
ect
ion l
i
mit
,pot
ent
ial
l
yreduci
ng

det
ect
iont
imeandi
mpr
ovi
ngdi
agnost
icqual
i
ty.PCR-
ELI
SAhasbeenpr
oposedf
oruse

i
nav
ari
etyofdi
sci
pli
nes,r
angi
ng f
rom si
mpl
edet
ect
ionanddi
agnosi
sto qual
i
ty

cont
rol
,
quant
it
ati
veobser
vat
ionofi
nfect
iousdi
sease,
andbi
omar
ker
s.Despi
tei
tshi
gh

speci
fi
cit
yandsensi
ti
vi
ty,t
her
ehav
ebeenongoi
ngef
for
tsi
nor
dert
ofur
theri
mpr
ove
anddev
elopt
heappl
i
cat
ionsofPCR-
ELI
SAi
nthenearf
utur
eforbet
tercl
i
nical
diagnosi
s.

Knowi
ngt
hisf
actt
hatcanceri
samal
i
gnantdi
sor
dert
hatcanbecausedduet
opoi
nt

mut
ati
onsi
nanyoft
hemul
ti
plegenesi
nvol
vedi
ncel
lcy
cle,dev
elopi
ngabet
terPCR-

ELI
SAt
obeabl
etodet
ectmul
ti
plesequencesatat
imewi
thoutcr
oss-
react
ivi
tywi
l
lgi
ve

abet
teri
nsi
ghti
ntot
het
reat
mentpl
anst
opr
emal
i
gnantcancerpat
ient
s.I
nthi
sar
ti
cle,

af
undament
ali
ntr
oduct
ionoft
het
echnol
ogyandi
tsappl
i
cat
ionsar
epr
esent
ed.

Key
wor
ds: Pol
ymer
ase chai
nreact
ion-enzy
me l
i
nked i
mmunosor
bentassay
,

deoxy
ribonucl
eot
ide,
str
ept
avi
din,
semi
quant
it
ati
ve,
ant
i-
fl
uor
escei
nant
ibodi
es

Li
stofabbr
evi
ati
ons:

PCR-
ELI
SA: Pol
ymer
asechai
nreact
ion-enzy
mel
i
nkedi
mmunosor
bentassay

DI
G: Di
goxi
geni
n

qPCR: quant
it
ati
vePol
ymer
aseChai
nReact
ion

f
g: Femt
ogr
ams

pg: Pi
cogr
ams

ng: Nanogr
ams

DNA: Deoxy
ribonucl
eicaci
d

RNA: Ri
bonucl
eicaci
d

I
ntr
oduct
ion:

I
twasi
n1989,
thatCout
eeetal
.dev
elopedanoni
sot
opi
chy
bri
disat
ionassayt
hatcoul
d
quant
if
yPCR-
ampl
i
fiedDNA i
nHI
V-1(
Humani
mmunodef
ici
encyv
irust
ype1)usi
ng

appr
opr
iat
elyl
abel
edRNA-pr
obe,t
hist
echni
quewascal
l
edPCR-
ELI
SAorPol
ymer
ase

chai
nreact
ion-enzy
mel
i
nkedi
mmunosor
bentassay
.Amonocl
onal
ant
ibodyt
o X174

DNA-
RNAhy
bri
dswasusedbyt
hem t
odet
ectspeci
fi
cRNAf
orbet
tercl
i
nicaldi
agnosi
s

[
1].PCRorpol
ymer
asechai
nreact
iondev
elopedbyKar
yMul
l
isi
n1983i
sst
il
li
nusef
or

cl
i
nical
diagnosi
sbutduet
oit
slowdet
ect
ionl
i
mitandot
hermet
hodcompl
ement
aryf
or

f
inal
resul
tmakei
tacumber
someandl
essef
fi
cientmet
hodascompar
edt
oPCR-
ELI
SA

[
2].Combi
ningPCRwi
thi
mmunol
ogi
cdet
ect
ionofspeci
fi
cDNAsequencesgi
veshi
gher

speci
fi
cit
yandbet
terr
eassoci
ati
onofhy
bri
dofnucl
eicaci
ds.

PCR-
ELI
SA empl
oyst
he i
mmobi
l
izat
ion ofbi
oti
nyl
ated DNA on a mi
cropl
ate and

quant
if
iest
hedesi
redDNApr
oduct
sampl
i
fiedbyPCR.Thest
epsi
nvol
veampl
i
ficat
ion,

i
mmobi
l
izat
ion,anddet
ect
ion.I
nit
ial
l
ytheDNAofi
nter
esti
sampl
i
fiedusi
ngPCRi
nthe

pr
esenceofl
abel
l
eddeoxy
ribonucl
eot
ide(
usual
l
yiti
sdi
goxi
geni
n-11-
dUTP(
DIG-
dUTP)
)

.ThesePCRpr
oduct
sthenar
emadet
obi
nda5’
-
biot
inl
abel
l
edpr
obet
hathel
psi
nthe

i
mmobi
l
izat
ionoft
heDNA-
probehy
bri
dtot
hemi
cropl
ateusi
ngst
rept
avi
dincoat
edi
n

t
hewel
l
s.Theav
idi
n-bi
oti
ncompl
exi
mmobi
l
izest
hespeci
fi
cDNAofi
nter
estont
othe

pl
ate,
andr
emov
alofundesi
redpr
oduct
soccur
sdur
ingwashi
ngst
eps.Thequant
it
ati
ve

est
imat
ioni
sfi
nal
l
ydonebyaddi
ngant
ibody
/conj
ugat
e(ant
i-
DIG-
per
oxi
daseconj
ugat
e,

f
orexampl
e)whi
char
emadet
oconv
ertasubst
rat
e(2,
2′
-azi
no-
di-
3-et
hyl
benzt
hiaz
oli
ne

sul
fonat
e(ABTS)i
ncaseofant
i-
DIG ant
ibody
)toacol
oredpr
oduct
.Si
nce1989,a

numberofadv
ancement
shav
ebeenmadet
oimpr
ovet
hesensi
ti
vi
tyandspeci
fi
ci
tyof

t
hist
echni
quesot
hati
tsappl
i
cat
ioncanbespr
eadt
ovar
iousbi
ologi
cal
fiel
ds.
 Fi
g1.Basi
cSchemeofPCR-
ELI
SAt
echni
que

Theonl
ydr
awbackofusi
ngt
hist
echni
quef
ornewernucl
eicaci
dsequencesort
arget

genecont
aini
ngsampl
esi
sthatunt
ilt
hesequencei
snotknown,
probesand/
orpr
imer
s
can'
tbebui
l
t.

Appl
icat
ions

Themai
nbenef
itofusi
ngt
hist
echni
quei
sthati
tcanbeusedf
orassay
ingmul
ti
-panel

sampl
est
husl
argescal
escr
eeni
ngcanbedoneaf
teropt
imi
zat
ion.PCR-
ELI
SAasa

di
agnost
icanddet
ect
iont
echnol
ogy
,hasbeenef
fect
iveduet
oit
sexcel
l
entspeci
fi
cit
y

andsensi
ti
vi
ty.I
nmedi
caldi
agnost
ics,i
tisusedt
oident
if
yar
angeofai
l
ment
sand

i
nfect
ions,i
nfect
ionscaused byi
nvasi
vef
ungii
nimmunocompr
omi
sed i
ndi
vi
dual
s,

det
ect
ion ofpol
i
ovi
rus,ent
erov
irus,and nor
ovi
ruset
c.[
2]I
nthef
ood sect
or,t
his

t
echni
que has been used t
o i
dent
if
y hazar
dous f
ood-
bor
ne mi
croor
gani
sms;

Campy
lobact
erspp.
,Sal
monel
l
a,Li
ster
iamonocy
togenesandE.col
i
,ar
eonl
yaf
ew

exampl
es.I
nbot
hwat
ersuppl
yandi
ndust
ri
alcool
i
ngsy
stem wat
er,PCRELI
SAcan

i
dent
if
y t
he pr
esence of haz
ardous wat
erbor
ne mi
croor
gani
sms. Wat
erbor
ne

mi
croor
gani
smsi udeEscher
ncl ichi
acol
i
,Ci
tr
obact
erspp.
,Ent
erobact
eraer
ogenesand

Kl
ebsi
ell
a spp.
Asar
api
dindi
cat
iveassay
,PCR-
ELI
SA canbedonef
orquant
it
ati
ve

moni
tor
ing.Sev
eralr
esear
chesal
soi
ndi
cat
e,usi
ngt
het
estf
orquant
it
ati
vemoni
tor
ing

asar
api
dindi
cat
oroft
hei
ncl
usi
onorexcl
usi
onofaspeci
fi
csubst
rat
e,aswel
lasi
ts

pr
edi
ctedquant
it
y.

Ther
ear
eanumberofmi
croor
gani
smsdet
ect
edbyPCR-
ELI
SAf
orv
ari
ouspur
poses.

● Tr
ypanosomes[
3]-

TheELI
SAf
ormatwasut
il
isedt
odet
ectahy
bri
dizat
ionsi
gnalut
il
isi
ngaPCRt
arget
ing
t
he18Sr
ibosomalgeneandsubsequentanal
ysi
swi
tht
rypanosome-
speci
fi
ccapt
ure

pr
obes.The18Sr
egi
onwasusedi
nthenewPCR-
ELI
SAt
est
,butt
hef
orwar
dpr
imerhad

t
obeadapt
ed.
Thenov
elpr
imerpai
rswer
eubi
qui
tousandef
fect
ivel
yampl
i
fiedDNA

f
rom av
ari
etyofspeci
es,i
ncl ngT.congol
udi ense,T.br
uceibr
ucei
,andT.v
ivax.A

pr
operassessmentoft
hePCR-
ELI
SAwi
tht
hePCRt
echni
quei
ndi
cat
edt
hatt
hel
att
er

t
estwasabout100t
imesl
essef
fect
ivei
ndet
ect
ingT.ev
ansit
hant
hePCR-
ELI
SA.

Fi
g2.Speci
esspeci
fcTr
i ypanosomedet
ect
ionf
rom bl
oodsampl
e

PCR-
ELI
SAgenot
ypi
ngt
echni
quet
odet
ectr
ecessi
vedi
sor
der
s.

● Bet
a-t
hal
assemi
a[4]

β-
Thal
assemi
aiscausedbyr
educed(
β+)orabsent(
β0)sy
nthesi
soft
heβ-
globi
nchai
ns

of hemogl
obi
n.I
t pr
oduces sev
ere anaemi
a and necessi
tat
es f
requent bl
ood

t
ransf
usi
ons,
whi
chcanl
eadt
opr
obl
emsi
ncl
udi
ngi
ronexcessandal
l
oimmuni
zat
ion.I
n

compar
ison t
o exi
sti
ng t
echni
ques (
DNA sequenci
ng and ARMS)
,the PCR-
ELI
SA
genot
ypi
ngsy
stem wasabl
etodet
ectt
hef
ourmostf
requent(
almost60%)bet
a-

t
hal
assemi
apoi
ntmut
ati
ons(
IVS-
II
-1(
G-A)
,IVS-
I-
5(G-
C),
FSC8/
9(+G)
,andI
VS-
I-
110(
G-

A)
.DNAwasext
ract
edf
rom whol
ebl
ood,andDI
G-l
abel
i
ngPCRwasusedt
oampl
i
fya

por
ti
onoft
hebet
a-gl
obi
ngene.TheDI
G-l
abel
edPCRampl
i
conswer
edenat
uredand

combi
nedwi
thbi
oti
nyl
atednor
malandmut
antpr
obes(
fornor
malandmut
antgene

al
l
eles,
respect
ivel
y).Col
ori
met
ri
cELI
SAwasusedt
odet
ectt
hehy
bri
ds.
 Fi
g3.Bet
aThal
assemi
amut
ati
ondet
ect
ionusi
ngPCR-
ELI
SA

Scr
eeni
ngofmi
crobesi
nor
dert
oassur
efoodsaf
ety

● E.col
i[5,
6,
7]

Toi
dent
iyEscher
f ichi
acol
iO157:
H7andot
herShi
gat
oxi
n-pr ngE.col
oduci i(STEC)i
n

f
oodsampl
esi
ncl
udi
nggr
oundbeefandr
awmi
l
k,asensi
ti
veandspeci
fi
cPCR–ELI
SA

wasdev
eloped.MostPCRt
est
suset
woset
sofpr
imer
s(mul
ti
plex)t
hatar
especi
fi
cfor

st
x1andst
x2,
respect
ivel
yfordet
ect
ingSTEC.

● Lact
icaci
dbact
eri
a[8]

Fort
hescr
eeni
ngofl
argenumber
sofbact
eri
ali
sol
atesf
rom f
erment
edv
eget
abl
es,

PCR–ELI
SAshowedt
obeanef
fect
ivemet
hod.Thi
swi
l
lbehel
pfuli
nident
if
yingst
rai
ns

t
hatar
eidealf
ort
hedesi
gnofst
art
ercul
tur
esf
ort
hef
erment
ati
onofpl
antmat
eri
al.

Lact
obaci
l
li
,the Leuconost
oc f
ami
l
y, Lact
obaci
l
lus pent
osus/
plant
arum, and

Lact
obaci
l
lusbr
evi
swer
eal
lident
if
iedusi
ngol
i
gonucl
eot
idepr
obesf
orhy
bri
dizat
ioni
n

PCR–ELI
SA.A col
or-
dev
elopi
ngr
eact
ionwasempl
oyedt
odet
ectt
hehy
bri
des.The

Lact
obaci
l
lus-
speci
fi
c pr
obe l
ab876 was used i
n PCR–ELI
SA t
o qui
ckl
y scr
een

l
act
obaci
l
lif
rom sauer
kraut
, and t
he speci
es-
speci
fi
c pr
obes l
ab448 (
Lact
.

pent
osus/
plant
arum)andl
ab86wer
eusedt
ofur
therdi
ff
erent
iat
ethespeci
es.

● Sal
monel
l
aty
phi[
9]

Foodbor
nebact
eri
alpat
hogensar
ethemostser
ioust
hreatt
ofoodsaf
etybecauset
hey
ar
eabundant
lyspr
eadacr
osst
hewor
ld,i
nfl
i
cti
ngt
housandsofhumani
nfect
ionsand

economi
closseseachy
ear
. Forexampl
e,f neSal
oodbor monel
l
aspp.ar
ezoonot
ic

f
oodbor
ne bact
eri
athatcan cause f
ood poi
soni
ng.A PCR-
ELI
SA t
echni
que was

i
ntr
oducedt
otar
getspeci
fi i
cgenes,nv
AofSal
monel
l
aspp.i
nfood.

Di
agnosi
sofv
irusi
ncl
i
nical
sampl
es

● Humanpapi
l
lomav
irus(
HPV)[
10]

I
nfect
ionswi
tht
hehumanpapi
l
lomav
irus(
HPV)ar
eli
nkedt
oav
ari
etyofcut
aneousand

mucosaldy
spl
asi
as,i
ncl
udi
ngbot
hbeni
gnandmal
i
gnantl
esi
ons.PCR–ELI
SA,ont
he

ot
herhand,
maybeusedt
odet
ectHPVgenomesandt
oident
if
yvar
iousv
iralgenot
ypes

usi
ngt
ype-
speci
fi
cpr
obes,andi
thast
headv
ant
ageofhi
ghsensi
ti
vi
tyofenz
yme

ampl
i
ficat
ionandspeci
fi
cit
yoft
hehy
bri
dizat
ionr
eact
ion.

● Humani
mmunodef
ici
encyv
irust
ype1(
HIV-
1)[
11,
12]

Humani
mmunodef
ici
encyv
irust
ype1(
HIV-
1)i
nfect
ionoft
hecent
ralner
voussy
stem

causesneur
odegener
ati
onanddement
ia,
whi
char
efr
equentAI
DSconsequences.

Fort
hedet
ect
ionofHI
V-1,PCR-
ELI
SAhasbeenshownt
obesensi
ti
veandspeci
fi
c.I
n

st
udi
esi
n2009and2013i
nIr
an,t
hePCRELI
SAmet
hodwascompar
edt
othenest
ed-

PCRmet
hod.Asaconcl
usi
on,
PCR–ELI
SAout
per
for
mednest
ed-
PCRbyaf
act
orof10.

Advant
agesoverot
hert
echni
quesi
nmol
ecul
ardi
agnosi
s
Thi
smet
hodsi
ncei
t'
sdev
elopmenthasbeenunder
goi
ngcont
inuousi
mpr
ovementand

i
sbet
tert
obeusedf
orcl
i
nicalsampl
est
hatot
her
wisewoul
dbecont
ami
nat
edwi
th

mut
agen-
stai
ningcompounds.Usi
ngpr
obest
hatcor
respondt
oorar
ecompl
ement
ary

t
othesampl
efordet
ect
ingpar
ti
cul
argenet
icsequences,
giv
esi
tbet
terspeci
fi
cit
ythan

met
hods l
i
ke el
ect
rophor
esi
s orbl
ott
ing t
echni
ques [
2].Al
so when compar
ed t
o

conv
ent
ionalPCR,
itgi
vest
estr
esul
tsi
nlessert
imegi
vi
ngadv
ant
aget
opat
ient
sunder

sev
ereand/
orundi
agnosedcondi
ti
on.Duet
oit
ssemiquant
it
ati
venat
ure,
agener
ali
dea

ofpr
ogr
essi
onofadi
seasecanbeobt
ained.

PCR-
ELI
SA i
sa si
mpl
etechni
que t
hatdoesn'
trequi
reheav
ymachi
nesordi
ff
icul
t

anal
yti
calequi
pmentf
oranal
ysi
ngt
hesampl
e.Thushi
ghexper
ti
sei
snotr
equi
redf
or

t
heconduct
ionofexper
iment
s.Addi
ngt
oitt
heuseofspeci
fi
cDNA/
RNApr
obesf
or

i
mmobi
l
izat
ionhel
pdi
sti
ngui
shspeci
esofbact
eri
aorot
hermi
croor
gani
smsbasedon

t
he speci
fi
c sequences excl
usi
vet
othe or
gani
sm.PCR-
ELI
SA i
s 10 t
imes mor
e

sensi
ti
vet
hannest
ed-
PCRduet
olowerr
iskofcont
ami
nat
ingagent
sandav
oidanceof

f
alsenegat
iver
esul
tsi
ncaseofl
owv
iral
load[
13]
.
 Fi
g4.Adv
ant
agesofPCR-
ELI
SA

Thepr
osoradv
ant
agesofusi
ngPCR-
ELI
SAf
orcl
i
nical
labt
est
sar
e:

● Col
ori
met
ri
ctestwi
thbet
terspeci
fi
cit
y

Si
nceasubst
rat
e-enzy
mer
eact
ioni
susedt
opr
ovi
decol
ort
othesampl
eint
he

wel
l
s,col
ori
ntensi
tygi
ves pr
opor
ti
onalr
esul
tst
othe speci
fi
c nucl
eic aci
d

amount
.Thi
s,i
naway
,gi
vessemi
quant
it
ati
veest
imat
ionandani
deaofsev
eri
ty

or pr
ogr
essi
on of di
sease or an appr
oxi
mat
e l
oad of speci
fi
c gene

(mi
croor
gani
sm i
ncaseofcl
i
nical
diagnosi
s)i
nthesampl
e.

● Less t
ime consumi
ng t
han onl
y PCR and mi
croscopi
c or ot
hercul
tur
al

t
echni
ques

Cul
tur
ingbact
eri
arequi
resshaki
ngoft
hei
nocul
um i
nani
ncubat
orshakerf
or

aboutov
erni
ghtwhi
l
ePCR-
ELI
SAcanbeconduct
edandcompl
etedwi
thi
nar
ound

4hour
s[14]
.Det
ect
ingder
mat
ophy
tesi
sbasedonmi
croscopi
cdet
ermi
nat
ion

andcul
tur
etest
ingwhi
chi
sti
meconsumi
ngandt
akesmor
ethan24hr
s.Asper

ast
udy
,isol
atedgenomi
cDNAofski
nscr
api
ngsandnai
lsampl
esf
rom pat
ient
s

wast
est
edusi
ngPCR-
ELI
SA(f
orspeci
esspeci
fi
ctopoi
somer
aseI
Igene)
,PCR

onl
yandcul
tur
egr
owt
hinf
iveTr
ichoder
maspeci
es.Ther
esul
tsshowedt
hat

onl
y25% ofsampl
eswer
edecl
aredposi
ti
veusi
ngPCrandonl
y7.
3% posi
ti
ve

wi
thcul
tur
etest
ing,whi
l
ePCR-
ELI
SA wi
thhi
ghsensi
ti
vi
tyandspeci
fi
cit
yfor

speci
esgav
ethebestr
esul
ts[
15]
.Anot
herst
udyt
odet
ectcol
i
for
msi
ndr
inki
ng

wat
eri
nvol
vedusageof16Sr
RNApr
obesi
nPCR-
ELI
SAt
odi
ff
erent
iat
ebet
ween
 Escher
ichi
acol
i
,Ci
tr
obact
erspp.
,Ent
erobact
eraer
ogenes,andKl
ebsi
ell
aspp.

usi
ng met
hods l
i
ke mul
ti
ple-
tube f
erment
ati
on, membr
ane f
il
tr
ati
on,

i
mmunof
luor
escence ant
ibodi
es (
IFA)
,chr
omo agarcul
tur
e medi
atest
,and

f
luor
escence i
n si
tu hy
bri
dizat
ion (
FISH)ot
hert
han PCR-
ELI
SA.The r
esul
ts

showedt
hatPCR-
ELI
SAhasbet
tersensi
ti
vi
ty,
LOD,
accur
acyandl
owerdet
ect
ion

t
ime[
14]
.

● Bet
tersensi
ti
vi
tyf
ordet
ermi
ningi
nfect
ionr
ate

Accor
ding t
o ast
udyi
n Sr
iLanka,compar
i he Wucher
ng t eri
abancr
oft
i(W.

bancr
oft
i)i
nfect
ionr
atesofCul
exqui
nquef
asci
atust
hatcancausel
ymphat
ic

f
il
ari
asi
s,wasdoneusi
ngPCR-
ELI
SAanddi
ssect
ionst
udi
es,r
eveal
i
ngt
hatt
he

pr
eval
ence r
ate was bet
tershown usi
ng PCR-
ELI
SA t
han bydi
ssect
ing t
he

mosqui
toest
odet
ermi
net
hel
arv
ae[
16]
.

● Si
mul
taneousdet
ect
ionofmul
ti
plesampl
e

Mul
ti
plei
sol
ates can be di
ff
erent
iat
ed i
n a si
ngl
etestbyusi
ng pr
imerf
or

di
ff
erentr
egi
onsoft
hegenome,bei
tcodi
ngornon-
codi
ngr
egi
onsspeci
fi
ctoa

speci
es.Ti
l
lti
mesev
eralbact
eri
aland v
iralspeci
esofasi
ngl
egenust
hat

happent
obei
nfect
ioushav
ebeeni
sol
atedanddi
ff
erent
iat
edf
rom t
estsampl
es.

Aswasev
identf
rom ar
esear
chf
ocussi
ngondi
ff
erent
iat
ingFMDV(
Foot
-and-

Mout
hdi
seasev
irus)ser
oty
pesO,A,andAsi
a1usi
ngmul
ti
plexRT-
qPCRassay

usi
ngpr
obet
arget
ingFMDVVP1codi
ngr
egi
on[
17]
.
 ● Gr
eatsensi
ti
vi
tyandbet
terl
i
mitofdet
ect
ion

Thesensi
ti
vi
tyandl
i
mitofdet
ect
ionofPCR-
ELI
SAassayhasbeendet
ermi
nedt
o

06×102and1.
be1. 06×103CFU/
mlf
orpur
ecul
tur
eswi
thandwi
thoutenr
ichment
.

Thel
owestconcent
rat
iont
hatcoul
dbedet
ect
edwasi
nfemt
ogr
amsperµl
.

Theunder
lyi
ngt
abl
eindi
cat
est
heor
gani
smsandt
hei
rdet
ect
ionl
i
mitusi
ngPCR-

ELI
SA [
13]

Or
gani
smsdet
ect
edbyPCR-
ELI
SA Det
ect
ionl
imi
t

Schi
stosoma 1.
3fg/
µl

E.col
iO157:
H7 1.
08pg/
µl

Shi
gel
l
a 1.
56pg/
µl

HAVorHEV 0.
1ng/
µl

Kl
ebsi
ell
apneumoni
a 0.
62ng

Vi
bri
ochol
eraO1 0.
5pg/
µl

Tabl
e1.Thepr
eci
sedet
ect
ionl
imi
tofsomemi
croor
gani
smsdet
ermi
nedusi
ng

PCR-
ELI
SA

Advancement
sint
het
echni
que

Si
ncei
tsdev
elopment
,manyadv
ancement
shav
ebeenmadei
nthi
stechni
que,
basi
cal
l
y
f
ocussi
ngont
hei
mmunodet
ect
ionpar
t.Usi
ngf
luor
escentpr
obesl
i
kef
luor
escei
nand

ant
i-
fl
uor
escei
nant
ibodi
esconj
ugat
edt
ohor
ser
adi
shper
oxi
dase(
HRPO)f
ordet
ect
ion

ofl
abel
l
ednucl
eicaci
discommonnow [
2].Oneoft
her
ecenti
mpr
ovement
sint
his

t
echni
quehasbeent
hedev
elopmentofamul
ti
plexi
ngsy
stem f
ordet
ect
ionofmul
ti
ple

sequencesi
nonegowi
thoutcr
oss-
react
ivi
ty.Theneedf
ort
hiswast
obeabl
etodet
ect

mul
ti
plespeci
esatt
hesamet
imeandt
omakei
tcost
-ef
fect
ive.

Anot
her dev
elopment appr
oach was t
o dev
elop asy
mmet
ri
c PCR-
ELI
SA whi
ch

el
i
minat
edt
her
equi
rementf
ordenat
urat
ionandneut
ral
i
zat
ionofnucl
eicaci
dsampl
es

bef
orei
mmobi
l
izat
ionont
othemi
cropl
ate.Thi
sinv
olv
edampl
i
ficat
ionofonl
yone

st
randofdoubl
est
randedDNAbyusi
ngpr
imer
sfort
hedesi
redst
randsequence.Thi
s

notonl
yreducedt
het
imeandcostofl
essdNTPsandl
abel
sbei
ngusedbutal
so

i
ncr
easedt
heconcent
rat
ionoft
arget
edDNAspeci
es[
18]
.

Anot
herappr
oacht
oel
i
minat
ethest
epofpr
obi
ngDNAwasdonewhi
l
edet
ect
ingand

quant
if
yingLei
shmani
apar
asi
tesbyusi
ngbot
hDI
G-andBi
oti
n-l
abel
l
edpr
obesf
orPCR

ampl
i
ficat
ionst
ep.Theampl
i
fied pr
oduct
swer
ethenat
tachedt
othest
rept
avi
din-

coat
edpl
atebef
oret
hepr
oduct
scoul
dbedet
ect
edusi
ngsandwi
chELI
SAusi
ngant
i-

DI
Gant
ibodi
es[
19]
.

Ot
heri
mpr
ovement
shav
ebeent
heuseofdi
ff
erentt
ypesofPCRl
i
keRT-
PCRandq-
PCR

coupl
edwi
thELI
SAt
oincr
easet
hesensi
ti
vi
tyoft
het
echni
que.Aswasev
identf
rom a

r
esear
chf
ordi
ff
erent
iat
ingHAV(
Hepat
it
isAv
irus)f
rom HEV(
Hepat
it
isEv
irus)
,dupl
ex-

RT-
PCRwasabl
etodet
ectasl
i
ttl
eas0.
1ng/
LHAVandHEVi
nthesampl
e[20,
21]
.

Fort
hedet
ect
ionofhumanv
iscer
all
eishmani
asi
s(HVL)
,akDNAPCRenzy
me-
li
nked

i
mmunosor
bentassay(
kDNA PCR-
ELI
SA)wasdev
eloped.ThekDNA PCR-
ELI
SA i
s
based on t
hecapt
ureofan ampl
i
fied pr
oducti
n apol
yst
yrenepl
atecoat
ed wi
th

st
rept
avi
din wi
th a sense pr
imerl
abel
l
ed wi
th bi
oti
n att
he 5′end,f
oll
owed by

hy
bri
dizat
ionwi
thaf
luor
escei
n-l
abel
edpr
obeatt
he5′endandanant
ifl
uor
escei
n-

per
oxi
daseconj
ugat
efordet
ect
ion.Becauseki
net
opl
astDNA,orkDNA,i
sabundanti
n

heLei
t shmani
acel
l
,itwaschosenast
heDNAt
argetf
ort
hePCR-
ELI
SA.ThekDNAPCR-

ELI
SAi
sapr
omi
singt
echni
quef
ori
dent
if
yingandf
oll
owi
nguponasy
mpt
omat
icpeopl
e,

especi
all
yinpopul
ati
on-
basedst
udi
eswi
thahi
ghnumberofpeopl
etoexami
ne.

LAMP (
loop-
medi
ated i
sot
her
malampl
i
ficat
ion)i
sat
echni
quef
orampl
i
ficat
ion of

nucl
eicaci
ds.LAMPhasal
sobeencoupl
edwi
thELI
SAbyi
ntegr
ati
ngant
igen-
label
ed

nucl
eot
idesi
ntoLAMPampl
i
const
hroughoutt
heampl
i
ficat
ionpr
ocess;t
hist
echni
que

i
sconsi
der
edasanot
hermodi
fi
cat
ionofPCRELI
SA.Thecapaci
tyt
opr
ocesshundr
eds

ofsampl
esconcur
rent
lyi
namat
terofhour
sist
hemaj
orbenef
itofLAMP-
ELI
SA.The

combi
nat
ionofLAMPandELI
SApr
ovi
desexcept
ional
l
yhi
ghspeci
fi
cit
y,al
l
owi
ngev
ena

si
ngl
ecopyoft
argetDNAt
obedet
ect
ed[
22]
.

Ot
hert
hant
hat
,compl
ement
ingmul
ti
plex-
RT-
PCR wi
thot
hert
echnol
ogi
esl
i
kesl
i
de

mi
croar
rayoraut
omat
edel
ect
roni
cmi
croar
rayassayf
ordet
ect
ingv
irusl
i
kesswi
ne

v
iruses,i
ncl
udi
ngFMDVandot
herv
irusessuchasSVDV,Af
ri
canswi
nef
everv
irus,

por
cineci
rcov
irust
ype2,por
ciner
espi
rat
oryandr
epr
oduct
ivesy
ndr
omev
irus,VESV,

andcl
assi
calswi
nef
everv
irus,cani
ncr
easet
hesensi
ti
vi
tyoft
het
echni
quebyonel
og

uni
t[23]
.

Open sandwi
ch PCR-
ELI
SA f
ordet
ect
ing smal
lmol
ecul
es orant
igens r
elat
ed t
o

di
seaseshasbeenusedf
ornoncompet
it
ivedet
ect
ionandquant
if
icat
ionbyut
il
izi
ng

ant
igen-
dependentst
abi
l
izat
ionofanant
ibodyv
ari
abl
eregi
on.Thedet
ect
ionpr
inci
plei
s
t
hedependenceofant
igenont
hei
nter
act
ionbet
weent
heheav
yandl
i
ghtchai
noft
he

v
ari
abl
eregi
on oft
he ant
ibody
.Thi
stechni
que has been used f
ordet
ect
ion of

ost
eocal
cin(
Bone Gl
a Pr
otei
n,BGP)usi
ng t
he r
ecombi
nantf
usi
on pr
otei
n .The

det
ect
ionl
i
mitobt
ainedwas100f
g/ml
[24]
.

PCR-
ELI
SAi
ncancerdet
ect
ion

Si
nce conv
ent
ionalELI
SA can not det
ect t
he mal
i
gnant gene (
biomar
ker
)in

pr
emal
i
gnantst
agesoft
umorduet
oit
slowundet
ect
abl
elev
els,
PCR-
ELI
SAst
andsasa

bet
teropt
ion.I
nmanycases,dur
ingt
hei
nit
iat
ionoft
umordev
elopment
,themal
i
gnant

cel
l
stendt
obur
st,r
eleasi
ngt
hei
rnucl
eicaci
dint
hebl
oodst
ream t
hatcanbedet
ect
ed

i
nthecel
lfr
eesy
stem-ser
um orur
ine.Butsi
ncePCR-
ELI
SAi
nvol
vesat
argetspeci
fi
c

appr
oach,knownsequencesoft
hebi
omar
kergeneorpr
otei
nsequencear
emost
ly

usedf
ordet
ect
ion[
25]
.

I
mmuno-
PCRorPCR-
ELI
SAhasbeenusedf
ordet
ect
ionofcol
orect
alcancer(
CRC)by

det
ect
ing t
he concent
rat
ion oft
elomer
ase gene whi
ch has been shown t
o be

si
gni
fi
cant
lyi
ncr
eased i
n case ofCRC [
26]
.Vi
ruses l
i
ke HPV hav
e pr
oven t
o be

associ
atedwi
tht
umor
soft
hemout
handl
ungst
hatf
urt
herl
eadt
oor
ophar
yngeal

cancer
s.Theyar
eimpl
i
cat
edmost
lyi
ntheor
alcav
ity
.HPVposi
ti
vet
umor
stendt
o

expr
esssomeconsensussequencei
ntheHPV genomef
orwhi
chpr
imer
scanbe

const
ruct
edanddet
ect
edusi
ngPCR-
ELI
SA[
27]
.

Butt
il
ltodayi
thas notbeen appr
oved f
orcancerdet
ect
ion and has st
ayed i
n

l
abor
ator
iesf
orr
esear
ch wor
k.I
tisbecause oft
hef
actt
hatcancergeneskeep

mut
ati
ngaspert
hecel
l
s’r
equi
rementandgr
owt
hinduci
ngchar
act
er.
Thef
oll
owi
ngt
abl
eshowst
hecancer
sthathav
ebeendet
ect
edusi
ngPCR-
ELI
SAand

t
hei
rrespect
ivebi
omar
kergenes[
25]:

Cancert
ype Bi
omar
kerdet
ect
edusi
ngPCR-
ELI
SA

Gast
ri
ccancer MG-
7Ag

Pr
ost
atecancer PSA

Br
eastcancer CEA,
CA15-
3,HER2+

Nasophar
yngeal
car
cinoma I
ggAant
ibodi
esagai
nstEBVcapsi
d

(
causedbyEpst
ein-
Bar
rvi
rus) ant
igen,
Ant
i-
EBVDNaseant
ibodi
es

Ov
ari
ancancer HB-
EGF(
Hepar
inbi
ndi
ng-
EGFl
i
kegr
owt
h

f
act
or)
,EGFL7

Bonecancer OPG

Tabl
e2.Somecommoncancer
sdet
ect
edusi
ngPCR-
ELI
SAandt
hei
rrespect
ive

bi
omar
kergenesusedi
nthet
echni
que

Concl
usi
on:

PCR-
ELI
SAi
sanov
elt
echni
quet
hati
shi
ghl
yspeci
fi
candmor
esensi
ti
ve(
atl
east10
t
imes mor
e sensi
ti
ve)t
han t
he conv
ent
ionalt
echni
ques t
hathav
e been used f
or

di
agnosi
sanddet
ect
ion.I
tsdet
ect
ionl
i
miti
sthef
emt
ogr
amsandi
sofconsi
der
abl
e

i
mpor
tancef
orsmal
lsampl
esi
zes.Themaj
orst
epi
sthei
nit
ialsy
nthesi
sofaspeci
fi
c

pr
imerwhose sequence i
staken f
rom NCBIand bl
ast
ed f
orcr
oss-
checki
ng t
he

speci
fi
cit
yofpr
imer
s.Useofdi
ff
erentpr
obeshasi
mpr
ovedt
hev
isual
i
zat
ionoft
he

sampl
ewi
tht
ime.I
tsappl
i
cat
ionsar
esl
owl
yspr
eadi
ngt
odi
ff
erentf
iel
dswi
tht
he

adv
ancement
sbei
ngbr
oughti
n.Thi
stechni
que,al
thoughbei
ngsemi
-quant
it
ati
ve,has

been used i
n det
ect
ion ofdi
ff
erentbact
eri
a,f
ungaland v
iralspeci
es and f
or

di
ff
erent
iat
ionbet
weencl
osel
yrel
atedspeci
esoft
hesamegener
a.PCRELI
SA has

pr
ogr
essedsi
gni
fi
cant
lyasar
esul
tofsev
eraladv
ances,
andi
tnowof
fer
sawi
dev
ari
ety

ofappl
i
cat
ions.I
ts appl
i
cat
ions ar
e sl
owl
y spr
eadi
ng t
o di
ff
erentf
iel
ds such as

env
ironment
almoni
tor
ing,
ident
if
yingcar
ri
ersf
orr
ecessi
vedi
sor
der
s,f
oodandnut
ri
ti
on

secur
it
yandaccur
atedi
agnosi
sofpat
hogeni
cbact
eri
aandv
iruses.Whencompar
edt
o

mol
ecul
art
echni
quesdependi
ng on gelel
ect
rophor
esi
s,PCR-
ELI
SA i
smuch mor
e

pr
eci
se,r
equi
resl
esst
ime,i
mpr
oveddet
ect
ionl
i
mitandmanysampl
esmaybet
est
ed

att
hev
erysamet
ime.

Fut
urepr
ospect
s:

I
mmuno-
PCRhasseensev
eraladv
ancement
sandi
sbei
ngusedf
orbet
terr
esul
tsi
nthe

cl
i
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