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

Abstract

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 ؋ 10 3 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 pointof-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 metho...

Gold Nanoparticles for DNA/RNA-Based Diagnostics

“…The system showed high sensitivity with a limit of detection (LOD) of 66 ng/mL of DNA. Following the same approach, several groups were able to develop methods for human immunodeficiency virus type 1 [9] and Cryptosporidium parvum [10] with direct detection capabilities, allowing to circumvent expensive enzymatic DNA amplification reactions. Recently, Weigum et al developed an amplification-free molecular assay for the detection of Cryptosporidium parvum oocysts [11].…”

A Promising Road with Challenges: Where Are Gold Nanoparticles in Translational Research?

“…These colorimetric-based assays with AuNPs have enhanced the detection sensitivity down to the femtomolar level of the target molecule, having tremendous potential for use at the point of care. The crosslinking aggregation methods have been extensively used in the last 10 years, especially for the DNA/RNA detection of microorganisms, such as M. tuberculosis and Bacillus subtilis spores [55,56], Cryptosporidium parvum [57], Chlamydia trachomatis [58], Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumonia, Serratia marcescens and B. cereus [59], Salmonella spp. [60], Neisseria gonorrhoeae, Treponema pallidum and Plasmodium falciparum [61], methicillin-resistant S. aureus [62], M. tuberculosis complex [62], virus-like human papiloma virus [63], Kaposi's sarcoma-associated herpes virus [64], HIV [56] and hepatitis B virus [61].…”

Gold Nanoparticles in Biomedical Applications

“…In one such study, oligonucleotides complementary to the unique sequences of the heat shock protein 70 (HSP 70) of Cryptosporidium parvum was used to functionalize GNPs, which could be used to detect the oocytes of Cryptosporidium in a colorimetric assay, offering a simple and robust method of molecular detection. 71 GNPs were used to detect Salmonella enteritidis and Listeria monocytogenes, where GNPs deposited within the flagella and in the biofilm network. 72 Similarly, GNP-Poly (para-phenyleneethynylene) could efficiently identify both Gram-positive and negative bacteria based on the differential response by each bacteria.…”