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Results
The nucleic acid lateral flow immunoassay
(NALFIA) for the simultaneous detection of the
pathogenic L. monocytogenes in particular and the
genus Listeria in general is using the combination of nucleic acid amplification and an immuno-
chemical based detection principle.FastStart
Taq DNA polymerase is a thermostable, modified
form of recombinant Taq DNA polymerase, with
which the occurrence of nonspecific amplification
products can be substantially reduced.PCR products from other
microorganisms (Bacillus, Enterococcus, Entero-
bacter, and Lactobacillus), and the primer control
(PCR without template DNA) were all negative
(Figure 3).
Results
The nucleic acid lateral flow immunoassay
(NALFIA) for the simultaneous detection of the
pathogenic L. monocytogenes in particular and the
genus Listeria in general is using the combination of nucleic acid amplification and an immuno-
chemical based detection principle. The detection
procedure starts with enrichment of the sample.
Following isolation of DNA a duplex PCR is per-
formed with two labelled primer sets. The PCR
solution is directly added to the one-step assay
device and the appearance of a grey/black line is
indicative of the presence of the specific amplicon.
Samples containing the L. monocytogenes specific
double-labelled amplicons were indicated by the
appearance of two grey/black lines; samples con-
taining the Listeria spp. specific double-labelled
amplicons were indicated by the appearance of
one grey/black line.
Duplex PCR for simultaneous amplification of
both generic Listeria spp. and specific L. mono-
cytogenes sequences was based on Herman et al..
(1995) and D’Agostino et al. (2004). Although these
PCR protocols were suitable for the detection of
amplicons on agarose gels (Figure 2), false positive
results were initially observed in the Listeria-NA-
LFIA. This problem was eliminated by decreasing
the amount of PCR cycles from 40 to 25 cycles and
by using Fast Start Taq DNA polymerase instead of
the conventional Taq DNA polymerase. FastStart
Taq DNA polymerase is a thermostable, modified
form of recombinant Taq DNA polymerase, with
which the occurrence of nonspecific amplification
products can be substantially reduced.
The specificity of the described NALFIA was
studied by testing a range of Listeria strains and
other food relevant microorganisms in artificially
contaminated milk samples. PCR products of all
tested L. monocytogenes strains bound with both
capture lines (the αDIG and the αFITC line). PCR
products of all other nonpathogenic Listeria only
bound the α-FITC line. PCR products from other
microorganisms (Bacillus, Enterococcus, Entero-
bacter, and Lactobacillus), and the primer control
(PCR without template DNA) were all negative
(Figure 3).
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