Utility of aptamer-fluorescence in situ hybridization for rapid detection of Pseudomonas aeruginosa

Pseudomonas aeruginosa, a ubiquitous Gram-negative bacteriuma, is considered one of the most important causes of nosocomial infections in immunocompromised patients with cancer, transplantation, burn or cystic fibrosis [1–4]. Rapid and accurate identification of P. aeruginosa has important implications for the therapy and management of infectious diseases caused by P. aeruginosa. Numerous new methods such as immunoassays and molecular techniques have been developed for its detection. However, these techniques usually go through several steps including isolation, enrichment and/or purification and require sophisticated equipment and highly trained personnel, which increase the assay time and cost [5, 6]. As a result, culture remains the gold standard. The requirement for a potent new method to identify P. aeruginosa accurately, rapidly and simply is obvious and compelling. It is well established that fluorescence in situ hybridization (FISH) is a highly valuable tool for the specific and rapid detection of pathogenic bacteria without cultivation [7]. For the purpose of P. aeruginosa identification, fluorescent antibodies bound to the outer membrane antigen are initially used as probes in FISH-bacterial detection [8]. Nowadays, such probes are rarely used in clinical practice for bacteria identification because it is hard to obtain a specific antibody without cross-reactivity and the labels on antibodies can cause them to loss their affinity to their target molecules. Recent studies have used DNA probes or peptide nucleic acid (PNA) probes to hybridizate with the intracellular components of pathogenic bacteria [9, 10]. However, it has been found that DNA-based FISH had limited value in clinical practice because separate fixation and/or permeabilization conditions are required dependent on the type of bacteria. Although without the prior enzymatic permeabilization steps in FISH assay, commercially available PNA probes are very expensive. So when attempting to design a new FISH, the question needs to be asked—What kind of probe is necessary? Aptamers are ssDNA or ssRNA oligonucleotides that can form stable and specific complexes with desired targets [11–14]. Through an in vitro process named SELEX (systematic evolution of ligands by exponential enrichment), aptamers binding to the targets with high selectivity and specificity are selected [15]. Aptamers are beginning to represent alternatives to antibodies in both therapeutic and diagnostic applications. In contrast to antibodies, aptamers are chemically stable, readily synthesized, and can be chemically modified and labeled more easily [16]. Many aptamer-based methods using aptamers as stationary phase or capture molecules have been established and can identify the whole bacteria in clinical and/or environmental specimens with improved or equivalent sensitivity and specificity of culture [17–19]. In FISH assay, DNA aptamers as probes for cancer cell identification and for protein localization in cells have been used [20, 21]. Although the potential value of aptamers as probes in FISH-bacterial detection has been mentioned in some academic articles [17], there is no such research available. And there is no work reported as to aptamers against P. aeruginosa for either diagnostic or therapeutic applications. In this study, we selected ssDNA aptamers specifically binding to P. aeruginosa based on a whole-bacterium Kai-Yu Wang and Yan-Li Zeng contributed equally to this work.