Biology Reference
In-Depth Information
A probe is an oligonucleotide (single stranded DNA or RNA)
with the size of 18 to 25 bases. The difference between probe and
primer is the function of the oligonucleotide. Primer is used during
polymerase chain reaction (PCR); however probe is used during
capturing of the target DNA during hybridization.
Short single-stranded synthetic targets (
20-60 bases oligonu-
cleotides) are used to evaluate the technology platform and
specificity of the DNA sequences in identification of bacteria before
the assay isevaluated with PCRamplicons.
The DNA duplex formation can be detected based on the
incorporation or association of a hybridization indicator or changes
accrued from the hybridization event. Different indicators can be
used in detection of DNA based on the appropriate electrochemical
activity selected, it can be either label-free (e.g. guanine, adenine),
or label-based (enzyme-based, ferrous and ferricyanide, Ruthenium
bipyridine [Ru (bpy)], methylene blue, Ethidium bromide etc).
The hybridization event is detected via the increase or decrease
in signal of the redox indicator or changes in conductivity or
impedance/capacitance.
Most genosensors are designed to detect bacterial DNA that
is first amplified by PCR. The species-specific detection of the
bacteria mainly depends on hybridization of the specific probes to
complementary sequences in the PCR amplicons. PCR primers and
hybridization probes are designed using bioinformatic softwares
to ensure high specificity and sensitivity. The bacterial genetic
sequences can be obtained from GenBank of National Center
for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). The
target genes selected will depend on the purpose of the test,
eitherforidentificationofinfectiousbacteria,detectionofantibiotic
resistant genotypes (MRSA, VRE, ESBL), bioterrorism (anthrax,
Burkholderia pseudomallei
or melioidosis,
Yersinia pestis
or plague),
food pathogens (
V. cholerae
,
Clostridium botulinum
,
Escherichia coli
0157:H7), fecal contamination (
Enterococcus
species) or environ-
mental surveillance. Then target gene sequences will be down-
loaded from Genbank and aligned by multiple sequence alignment
softwares available such as VectorNTI (Invitrogen Corporation,
California, USA). The highly specific gene regions (conserved
regions) willbe selected for primer designing.
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