Development of a Novel, Rapid Integrated Cryptosporidium parvum Detection Assay

Abstract

The aim of this study was to develop a reverse transcription-PCR assay and lateral flow detection protocol for specific identification of Cryptosporidium parvum. The method which we developed is sensitive and specific and has a low limit of detection. In our protocol a solid phase material, the Xtra Bind Capture System, was used for extraction and purification of double-stranded RNA (dsRNA) specific for C. parvum. The Xtra Bind Capture System interfaced with pellets concentrated from water samples collected with previously developed filtration devices. The pellets were resuspended in reagent water (final volume, 0.5 ml), and an equal amount of rupture buffer and the Xtra Bind Capture System was added to the resuspended pellet mixture. The dsRNA target sequences in a 0.5-ml portion were captured by the solid phase material via hybridization. The debris and potential inhibitors were removed by washing the Xtra Bind material several times with buffer. The Xtra Bind material with its bound dsRNA was added directly to an amplification reaction mixture, and the target was amplified without elution from the Xtra Bind material. A PCR was performed in the presence of the Xtra Bind Capture System, which resulted in robust amplification of the target. The detection system which we used was adapted from lateral flow chromatography methods typically used for antigen-antibody reactions. The result was a colored line that was visible if the organism was present. When this method was used, we were able to reproducibly and correctly identify 10 oocysts added to 0.5 ml of reagent water. When the protocol was evaluated with a small set of environmental samples, the level of detection was as low as 1 oocyst/liter. The total time from resuspension of the pellet to detection was about 3 h, which is considerably less than the 5 h required for immunomagnetic separation followed by an indirect immunofluorescence assay and microscopy.In recent studies workers found that Cryptosporidium parvum oocysts were present in 65 to 97% of the surface water tested at locations throughout the United States (29,30,36,37). Outbreaks have occurred in water systems ranging from simple chlorination systems to complete filtration and ozonation systems (5, 7, 9, 14, 15, 17-19, 32, 33, 38). An outbreak in Kitchner-Waterloo, Ontario, Canada, affected 1,000 people, and the water at this location was both filtered and ozonated. There have been more than 30 documented outbreaks of cryptosporidiosis in the United States. An outbreak during 1993 in Milwaukee, Wis., resulted in more than 400,000 cases of illness and 100 deaths (33). Filtration, which physically removes the parasite from contaminated water, is the only effective treatment since oocysts are resistant to chemical disinfectants. However, a filtration system that is not well maintained and operated may not provide absolute protection. Recent surveys in which the researchers examined the occurrence of Cryptosporidium oocysts in fully treated (disinfected and filtered) municipal water demonstrated that s...

Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey

“…A combination of antigen-antibody interaction and specific tagged doubled-stranded amplicon (ds-amplicon) detection after PCR is also possible, and is called "nucleic acid lateral flow immunoassay" (NALFIA) [39,40,42,69]. Specific nucleic acid hybridisation of amplicons with immobilised complementary probes is another option and is called "nucleic acid lateral flow assay" (NALF) [16,41].…”

“…In the NALFIA set-up the analyte is an amplified double-stranded nucleic acid sequence (dsamplicon) specific of the organism using primers with two different tags; recognition of the analyte is done by binding to a tag-specific antibody [39,40,42,69]. In a typical layout developed for the detection of pathogenic bacteria the nucleic acid was amplified using PCR with two tagged primers (Fig.…”

A Single-Tube Nucleic Acid Extraction, Amplification, and Detection Method Using Aluminum Oxide

“…A single tube nucleic acid extraction and amplification method based on embedded aluminum oxide and commercialized under the trade name Xtrana (Xtrana Inc., Broomfield, CO) has been previously described. 12 The Xtrana method has been used for extraction, amplification, and detection of nucleic acids as well as sequencing and reverse transcription. 13 The main limitation of the Xtrana method was suboptimal target sensitivity and detection, with observed crossing threshold lags of five to seven cycles (B. Linn, personal communication).…”

Carbon Nanoparticles as Detection Label for Diagnostic Antibody Microarrays

“…In this one-step format, the labelled amplicons were mixed with the conjugate of neutravidin and carbon nanoparticles in incubation buffer, immediately applied and detected after one to several hours. Such mixed immuno-DNA formats have been used in lateral flow and microfluidic detection assays (Baeumner 2004;Blazkova et al 2009;Blazkova et al 2011;Corstjens et al 2001;Koets et al 2009;Kozwich et al 2000;Mens et al 2008;Noguera et al 2011;van Amerongen & Koets 2005;Wang et al 2006). To get proof of concept for the use of carbon nanoparticles as signal labels in antibody microarrays we studied two applications in which the antigens consisted of double-tagged DNA amplicons: the detection of L. monocytogenes and the detection of three antibiotic resistance genes from Salmonella spp.…”