Oligonucleotide-Gold Nanoparticle Networks for Detection of Cryptosporidium parvum Heat Shock Protein 70 mRNA

We report on a novel strategy for the detection of mRNA targets derived from Cryptosporidium parvum oocysts by the use of oligonucleotide-gold nanoparticles. Gold nanoparticles are functionalized with oligonucleotides which are complementary to unique sequences present on the heat shock protein 70 (HSP70) DNA/RNA target. The results indicate that the presence of HPS70 targets of increasing complexity causes the formation of oligonucleotide-gold nanoparticle networks which can be visually monitored via a simple colorimetric readout measured by a total internal reflection imaging setup. Furthermore, the induced expression of HSP70 mRNA in Cryptosporidium parvum oocysts via a simple heat shock process provides nonenzymatic amplification such that the HSP70 mRNA derived from as few as 5 × 10³ purified C. parvum oocysts was successfully detected. Taken together, these results support the use of oligonucleotide-gold nanoparticles for the molecular diagnosis of cryptosporidiosis, offering new opportunities for the further development of point-of-care diagnostic assays with low-cost, robust reagents and simple colorimetric detection.The obligate intracellular protozoan parasites of the genus Cryptosporidium cause the diarrheal illness termed cryptosporidiosis (24). Approximately 30% of adults in high-income countries and nearly all adults in resource-poor countries have serologic evidence of prior infection with this organism. However, only a small minority of people have ever been diagnosed with clinical disease. This is thought to stem from the underutilization and poor sensitivities of current diagnostic tests. For example, in the outbreak in Milwaukee, WI, in which over 400,000 people developed diarrhea associated with contamination of the water supply with Cryptosporidium parasites, fewer than 1,000 cases were confirmed on the basis of stool examination (although nearly all individuals tested developed antibodies to the organism) (3). Studies employing sensitive assays (e.g., PCR) have greatly increased the ability to recognize the burden of cryptosporidiosis. For example, Cryptosporidium DNA was found in the stools of 20% of South African children with diarrhea and three-fourths of Ugandan AIDS patients with diarrhea (18, 22).The detection of oocysts in stool samples has served as the traditional basis for diagnosis. The organisms are usually overlooked by direct examination in the absence of special stains. Acid-fast stains (including modified the Ziehl-Neelsen and auramine O stains) or fluorescent dyes coupled to monoclonal antibodies (for the immunofluorescent-antibody test [IFAT]) are used in the standard assays used in clinical laboratories. The sensitivity of stool examination by acid-fast staining remains poor and requires an oocyst concentration of over 500,000 per ml in formed stools (23), with fewer cases being detected by acid-fast staining than by fluorescent methods (1, 12). IFAT has been reported to be up to 10 times more sensitive than acid-fast staining (5, 7, 10). Currently, the IFAT staining method is the “gold standard” for the microscopic examination of infected stool samples (2).Microscopy-based methods are increasingly being replaced with techniques that rely on molecular recognition for the specific targeting of the pathogens of interest. The enzyme-linked immunosorbent assay (13) and PCR (16) techniques have further improved the sensitivity and specificity of detection compared to those obtained by microscopic examination. However, these newer methods have limited applicability to point-of-care use or low-resource settings due to reagent and instrumentation costs, infrastructure needs, and the need for operator training. Rapid diagnostic tests for Cryptosporidium parasites, such as lateral-flow assays (8), have not been proven to be reliable alternatives to the existing methods.Recently, a simple colorimetric detection method that relies on the distance-dependent optical properties of oligonucleotide-functionalized gold nanoparticles has been described (6). This strategy offers a simple readout (a color change that can be visually monitored) and utilizes low-cost and robust reagents that have promising potential for point-of-care molecular diagnostics. Previous work has demonstrated that the color change associated with the target-induced formation of oligonucleotide-gold aggregates can be effectively monitored by spotting the solution on a glass slide illuminated via total internal reflection (20). The limit of detection of this approach for the detection of purified DNA from methicillin (meticillin)-resistant Staphylococcus aureus was previously shown to be <10⁵ bacteria (20).In the study described here, we demonstrate the detection of Cryptosporidium parvum oocysts using oligonucleotide-functionalized gold nanoparticles targeted against heat shock protein 70 (HSP70) DNA and RNA and this novel detection strategy. One advantage of targeting HSP70 is the ability to induce mRNA amplification by the use of a simple heating process that does not involve more complicated PCR-based amplification. We hypothesize that simple amplification (via the heat shock process) of targets present at very low copies (low zeptomole concentrations) in biological samples will facilitate the detection of the desired targets that are present at levels within the limit of detection of the gold aggregation assay.