Biology Reference
In-Depth Information
Thisstrategycanbeusedfortheelectrochemicalreal-timequan-
tification of amplicon since a linear relationship with the amount of
amplified product was obtained [75]. Moreover, this strategy is use-
ful only when a unique and specific band is observed by gel elec-
trophoresis,becauseofthehighspecificityofthesetofprimerbeing
used in the PCR for the amplification of the bacteria genome. On
the contrary, if the set of primers amplifies not only the sequence of
interestbutalsoothernon-specificfragments,itisnecessarytocon-
firm the internal sequence of the amplicon by a second hybridiza-
tion with a digoxigenin signaling probe. In this case, a single
labelingwithbiotinduringPCRwasperformedfollowedbyafurther
selective hybridization with a digoxigenin signaling probe. More-
over, magnetic bead primers were used for
in situ
amplification on
magnetic beads of the
Salmonella
genome and for further electro-
chemical detection of the amplified product. The DNA amplification
on magnetic beads by using the magnetic bead primer with electro-
chemical detection of the amplified product demonstrated to be an
alternative strategy to the classic detection systems. This strategy
wasalsoeasilyadaptedtoanimmunoseparationstepofthebacteria
to improve the LOD for detecting pathogenic bacteria[98].
The procedure consisted briefly of the following steps, as
schematicallyoutlinedinFig.3.6:(i)immunomagneticseparationof
the bacteria from food samples (Fig. 3.6A); (ii) lysis of the bacteria
and DNA separation (Fig. 3.6B); (iii) DNA amplification and double-
labeling of
Salmonella IS200
insertion sequence (Fig. 3.6C); (iv)
immobilization of the doubly labeled amplicon in which the biotin
extreme of the dsDNA amplicon was immobilized on the strepta-
vidin magnetic beads (Fig. 3.6D); (v) enzymatic labeling using as
enzyme label the antibody anti-DIG-HRP capable of bonding the
otherlabeledextremeofthedsDNAamplicon;(vi)magneticcapture
ofthemodifiedmagneticparticles;and(vii)amperometricdetermi-
nation [98].
In this approach, the bacteria are captured and preconcentrated
from food samples with magnetic beads by immunological reac-
tion with the specific antibody against
Salmonella
.Afterthelysis
of the captured bacteria, further amplification of the genetic mate-
rial by PCR with a double-tagging set of primers is performed to
confirm the identity of the bacteria. Both steps are rapid alter-
natives to the time-consuming classical selective enrichment and
