Biology Reference
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with digoxigenin. During PCR, not only the amplification of the
bacteria genome is achieved but also the double-labeling of the
amplicon ends with (i) the biotinylated capture primer to achieve
the immobilization on the streptavidin-modified magnetic bead,
and (ii) the digoxigenin signaling primer to achieve the electro-
chemical detection. Besides this double-labeling PCR strategy, a
single-labeling PCR strategy with a further confirmation of the
amplicon by its hybridization was achieved by performing the PCR
withthebiotinprimerandafurtherhybridizationstepwithadigoxi-
geninprobe.Theprocedureconsistsbrieflyofthefollowingsteps,as
schematically outlined in Fig. 3.5: (i) DNA amplification and double-
labeling of Salmonella IS200 insertion sequence; (ii) immobiliza-
tion of the doubly labeled amplicon in which the biotin extreme of
the dsDNA amplicon was immobilized on the streptavidin magnetic
beads (Fig. 3.5, A2); (iii) enzymatic labeling using as enzyme label
the antibody anti-DIG-HRP capable of bonding the other labeled
extremeofthedsDNAamplicon(Fig.3.5,B);(iv)magneticcaptureof
the modified magnetic particles (Fig. 3.5, C); and (v) amperometric
determination (Fig. 3.5, D) [97].
Moreover, a PCR reactor for real-time electrochemical detec-
tion was also designed (Fig. 3.5, A3). In this case, the amplifica-
tion and double-labeling is performed directly on the streptavidin
magnetic beads by using “magnetic bead primers” [97]. The proce-
dure consists briefly of the following steps: (i)
in situ
DNA amplifi-
cation and double-labeling of Salmonella IS200 insertion sequence
on streptavidin-modified magnetic beads by using a magnetic bead
primer (Fig. 3.5, A3); (ii) enzymatic labeling using as enzyme label
the antibody anti-DIG-HRP capable of bonding the other labeled
extremeofthedsDNAamplicon(Fig.3.5,B);(iii)magneticcaptureof
the modified magnetic particles (Fig. 3.5, C); and (iv) amperometric
determination (Fig. 3.5, D).
The rapid and sensitive verification of the PCR amplicon related
with
Salmonella
can be achieved with 2.8 fmol of amplified product
[97].Inthecaseof
E.coli
theassayshowedtobeverysensitive,being
abletodetect0.45ng
μ
l
−
1
oftheoriginalbacterialgenomeafteronly
10 cycles of PCR amplification [75]. Moreover, the electrochemical
strategies for the detection of the amplicon showed to be more sen-
sitive compared with Q-PCR strategies based on fluorescent labels
such as TaqMan probes.
