The gene or sequence that serves as target for the nucleic acids
detection needs to be defined, for instance through bioinformatics
analysis or database mining. A proper set of oligonucleotide probe
and hairpins must consequently be designed for, respectively,
binding and detecting the target sequence from the analyte
solution and then performing HCR amplification and confirmation
of the signal. For the experiments herein reported, the DNA
sequences of the necessary probes, targets and hairpins were
designed starting from sequences of target pathogenic microorganisms
(e.g. Cryptosporidium parvum) or viruses. The sequence of
a selected gene known to be specific for a strain, for example the
COWP gene in C. parvum, GenBank AF248743 (Yu et al., 2009), was
first screened automatically for potential candidate regions (for
good potential binding and low interfering secondary structure
and dimerization of the target). Then, a restricted number of
candidate sequence portions were individually checked in a
manual fashion to extract the candidate sequence with the lowest
predicted structural interference to HCR