Hybridization Chain Reaction Applications for Pathogens Detection in Solution

With the goal of determining the presence of pathogens when present in very small amount, such as for the screening of pathogens in drinking water, an analytical method of very high sensitivity must be implemented. As often this type of determinations should be performed at the point-of-care, with simple, automatic and inexpensive hardware, a chemical (or nanotechnological) signal-amplification step is required prior to biosensor read-out. The hybridization chain reaction (HCR), originally proposed by Dirks and Pierce (2004), is an isothermal, enzyme-free process where a hybridization event triggers the polymerization of oligonucleotides into a long albeit nicked double-stranded nucleic acid molecule. The two kinds of co-monomer oligonucleotides are present in solution as closed hairpins, are opened and added to the growing polymer only after the initial triggering hybridization event. We have designed HCR oligonucleotide sets to target DNA sequences of relevance for the protection from bioterrorism acts towards water systems. In this communication, we presents our results of the HCR amplifications for different DNA targets of C. parvum, G. lamblia and Hepatitis E virus that have been performed both in solution and in a microarray format, to make it more amenable of use in diagnostic or point-of-interest assays. Our need of detecting natural occurring sequences forcedly limits our possibility of sequence optimization, reducing it to the selection of a target sequence with low secondary structure within a consensus sequence defined by bioinformatics tools. The design of the HCR hairpins was the performed on the defined target sequences with the aid of NANEV (Goodman, 2005). Our results seem to point out that there is a general sensitivity advantage in a biosensor read-out of the presence of naturally occurring nucleic acids sequences when the hybridization chain reaction is used as a signal-amplification step. This strategy could result of general interest for the analytical determination of nucleic acids.