Book of Abstracts: Albany 2007
June 19-23 2007
Bacterial detection using PNA technology
A new approach is developed for fluorescent in situ detection of microbes. Highly specific recognition of target sequences in genomic DNA is achieved due to the use of peptide nucleic acid (PNA) openers followed by the oligonucleotide probe circularization on the displaced DNA strand (see cartoon in Fig. 1). A high sensitivity is achieved via rolling circle amplification (RCA). The approach allows the detection and identification of various bacteria on the basis of short signature sequences consisting of approximately 20bp and present in a single copy per bacterial genome. The proof-of-principle experiments have been performed on three bacterial species: Escherichia coli, Bacillus subtilis and Streptococcus mutans. Using different signature sites, reliable detection of all three bacteria was achieved. Using fluorophores with resolved spectra, simultaneous detection of two bacteria was also achieved. This newtechnique expands both the utility and resolving power of whole-cell FISH for the detection of microbes and may be useful in food, environmental and clinical diagnostics.
Based on this approach, we are developing this new method for diagnostic assays for Staphylococcus aureus, an important medical pathogen. The assay will detect S.aureus and at the same time reliably distinguish methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) strains in patient specimens.
Irina V. Smolina*
Figure 1. The PNA openers, clamp-like pyrimidine oligomers known as bisPNAs, have a unique ability to locally open double-stranded DNA (dsDNA) via binding to one of the two DNA strands leaving the other strand accessible for hybridization with synthetic oligonucleotide probes. After fixing bacteria on a slide and PNA binding, a circular probe is assembled by T4 DNA ligase, using a circularizable oligonucleotide with termini that are complementary to the displaced strand (step 2). Assembly of this circular probe is exceedingly sequence specific since only chosen sites are opened by PNA openers and the remaining DNA maintains its duplex form and is inaccessible for mismatch hybridization. Fluorescent labeling is achieved by a rolling-circle amplification (RCA) reaction on the circular probe in the presence of fluorescently-labeled decorators. These decorators consist of linear oligonucleotides with fluorophores at their termini; their hybridization to single-stranded DNA produced by RCA yields the multiply fluorescently-labeled product (step 3). The fluorescent signal is readily detected by standard techniques using fluorescent microscopy (step 4). (A ? C). Images of E. coli bacterial cells with the probes corresponding to the 21-nt-target site in E. coli cold shock protein gene region observed by fluorescent microscopy in experiments performed according to the proposed protocol directly in the human blood. Fluorescent signals were acquired separately using two filter sets: (A) DAPI only; (B) only Cy3 for the labeled RCA product; (C) is a superposition of two separate images, with DAPI and Cy3. Signals are pseudocolored in blue for DAPI and in red for Cy3.
References and Footnotes