Amplification-Free Detection ofCryptosporidium parvumNucleic Acids with the Use of DNA/RNA-Directed Gold Nanoparticle Assemblies

Abstract

This study describes the development and evaluation of an amplification-free molecular assay for detection of Cryptosporidium parvum oocysts. The assay employed a pair of oligonucleotide-functionalized gold nanoparticle (AuNP) probes that were complementary to adjacent sequences on C. parvum 18s rRNA. Hybridization of the probes to the target RNA resulted in the assembly of AuNPs into target-linked networks, which were detected both visibly and spectroscopically, by a red-shift in the wavelength of light scattered by the gold nanoparticles. The limit-of-detection was between 4 × 105 and 4 × 106 copies of RNA per μL reaction mix, when a short synthetic target or full length in vitro transcribed target were employed. With total nucleic acids purified from C. parvum oocysts spiked into 100 mg stool, as few as 670 oocysts/μL reaction mix were detected. The ability to detect the nucleic acids of C. parvum oocysts in stool, without the need for complex amplification, offers unique advantages for such AuNP aggregation assays to be extended toward use in resource-limited settings where protozoan detection is needed most.

Advancing Cryptosporidium Diagnostics from Bench to Bedside

“…The hybridization of nanoparticle probes to 18 s rRNA, which was done without amplification, was detected without complex technology was able to detect as few as 670 oocysts/ul in spiked stool samples. Cross-reactivity with either 18 s rRNA of other protozoan parasites or Escherichia coli was not observed [60]. Targeting the HSP70 gene, on the other hand, causes formation of oligonucleotide networks that are visually detected with simple colorimetric readout.…”

“…Quantum dot labels also have approximately 50 % lower interference in concentrated water samples compared to organic fluorophore labels [59]. Another applic a t i o n o f n a n o t e c h n o l o g y -b a s e d m a t e r i a l s f o r Cryptosporidium diagnosis is the use of oligonucleotide gold nanoparticles for molecular detection without the need for amplification of nucleic acids and proteins [60]. Examples of assays that have been developed using gold nanoparticles are the electrochemical-based sandwich enzyme-linked immunosensor [61], rapid immunodot blot assay [62], and amplification-free detection systems [60] by using a dual-labeled gold nanoparticles (alkaline phosphatase (ALP) and anti-oocyst monoclonal antibody) functionalized indium tin oxide electrode.…”

Molecular Medicines for Parasitic Diseases

“…The application of nanomaterials in the field of medicine for diagnosis and treatment received considerable attention in recent decades for parasitic diseases. The diagnostic potential of nanomaterials has been seen in malaria [106,107], toxoplasmosis [108], cryptosporiodiosis [109], amebiasis [110] and leishmaniasis [111,112].…”

Gold Nanoparticles for DNA/RNA-Based Diagnostics

“…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]. The assay targeted the C. parvum 18 s rRNA, with an LOD of 4 Â 10 5 copies of RNA per mL per reaction mix.…”