Biomedical Engineering Reference
In-Depth Information
Table 3 Primers and capture probes for the detection of PCR products in E. coli
Name Oligonucleotides and labeling (5
0
-3
0
)
vat f: GTGTCAGAACGGAATTGTC
#
, r: GGGTATCTGTATCATGGCAAG
p: (dT)
10
CCGGGGTTGCTTTATTTGAGAAATTAATATTTCCC
tsh f: TGGTGCAATGCCCATTTATGG, r: CTTCCGATGTTCTGAACGT
p: (dT)
10
TAATGAAGACAATGATGCCC
iucD f: CCTGATCCAGATGATGCTC
#
, r: CTGGATGAGCAGAAAATGACA
p: (dT)
10
TGGTCAGTAAAGAATCGGCAGTGATGCC
cvi/cva f: CGCAGCATAGTTCCATGCT
#
, r: GCAATTTGTTGCAGGAGGA
p: (dT)
10
TCATATATTGCACCTCCAGCCACACCCC
papC f: GCTCCATGGTCATATAGTTTCG, r: TGATATCACGCAGTCAGTAGC
p: (dT)
10
ACGTTCTCTCTCCCTCAATACG
iss f: GGACAAGAGAAAACTGTTGATGC, r: CAGCGGAGTATAGATGCCA
p: (dT)
10
CCAGTTATGCATCGTGCATATGG
EAST1 f: TGCCATCAACACAGTATATCC
#
, r: TAGGATCCTCAGGTCGCGAGTGACGGC
p: (dT)
10
CCAGTTATGCATCGTGCATATGG
The forward primer (f) was unlabeled, the reverse primer (r) contained a 5
0
-Cy5 label and the
capture probe (p) was 5
0
amino modified with an additional poly(dT)10 region. # oligonucleotide
sequence according to Ewers et al. [
62
]
E. coli for VAGs is multiplex-PCRs (mPCR) with subsequent agarose gel anal-
yses. This however is time-consuming, prone to errors and laborious. We therefore
developed a VideoScan multiplex hybridization assay with direct fluorescent
labeling of PCR products (see also [
52
,
61
]).
The mPCR was performed according to the following protocol: a 20-lL
reaction mixture was prepared including 2 lL109 PCR buffer, 2 lL50mM
MgCl
2
, 2 U Taq DNA polymerase (Rapidozym, Germany), 0.5 lL of each 10 mM
dNTP, 0.1 lL (100 pmol) primer pair (BioTez Berlin, Germany) (Table
3
), and 50
ng template DNA, supplemented with appropriate volumes of water. mPCR was
performed in a thermal cycler (Eppendorf, Germany): initial denaturation for 3
min at 94 C, 40 cycles of 30 s at 94 C, 30 s at 52 C, 1 min at 72 C, with a final
cycle of 5 min at 72 C. Hybridization of the fluorescence-labeled PCR fragments
to
the
immobilized
(
Sect.
3.1
)
capture
probe
(Table
3
)
loaded
microbeads
(
Sect. 3.2
) was achieved by hybridization for 120 min at 50 C.
The fluorescence intensity determined by VideoScan for each microbead pop-
ulation corresponded well with the amount of PCR product analyzed in parallel
with agarose gel electrophoresis (Fig.
14
). The analysis of three genes of E. coli
(iucD, cvi/cva, papC) by the VideoScan hybridization assay, in comparison to
standard agarose gel analysis, with a concentration gradation of template DNA
ranging from 0 to 50 ng is shown in Fig.
14
. The comparison of repetitive analysis
of seven VAGs of E. coli is shown as an example for three runs with three different
E. coli strains in Fig.
15
.
We have shown a multiplex hybridization assay for the detection of virulence-
associated genes (VAGs) as a sensitive diagnostic tool with a detection limit below
that of agarose gel band analysis. Besides the high sensitivity, it allows for very
specific mPCR analysis due to the exclusion of non-specific PCR products which
