Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR

Abstract

ABSTRACT:Cryptosporidium parvum is a common intestinal protozoan parasite infecting humans and a wide range of animals, whose diagnostics present considerable difficulties. These arise from the exceptionally robust nature of the oocyst's walls, which necessitates more stringent treatments for disruption and recovery of DNA for analysis using molecular methods. In the case of water, which is the major source of Cryptosporidium oocysts, investigations concern the detection of the presence of the oocysts. Their concentration in water is very low, and moreover, many substances that may have significance as inhibitors of DNA amplification, are present in environmental water and stool. We have carried out trials in order to assess the effectiveness of recovery of C. parvum oocysts, from spiked environmental and distilled water samples, filtrated and concentrated with the use of special laboratory equipment. Inactivation of inhibitors was carried out with use of bovine serum albumin (BSA) in PCR mixes at ten different concentrations. DNA extraction was carried out from stool samples spiked with C. parvum oocysts, concentrated using two methods, and unconcentrated. Nested PCR and a TaqMan nested real time PCR assay, targeting the 18S rRNA gene, was used to detect C. parvum DNA in spiked water and additionally in spiked stool samples. The obtained results showed that losses of C. parvum oocysts occur during the filtration and concentration of spiked water samples. The addition of small amounts of BSA (5-20 ng/µl) to PCR and TaqMan PCR mixes increases the sensitivity of both methods, but a high concentration of BSA (100 ng/µl and above) has an inhibiting effect on the polymerase reaction. The extraction of DNA from C. parvum oocysts from spiked stool samples preceded by concentration with PBS, ether and Percoll resulted in a higher copy number of the 18S rRNA gene.

High-Throughput Multiplex Quantitative Polymerase Chain Reaction Method for Giardia lamblia and Cryptosporidium Species Detection in Stool Samples

“…The sample was concentrated either by pelleting the oocysts by centrifugation followed by pellet resuspension or by sedimentation method that was based on sedimentation during a 1-hour incubation time in room temperature. 14,18 In the next step, different methods were compared, which disrupt the oocysts, such as exposure to combinations of proteinase K treatment, freeze-thaw cycles, and heat shock treatment (10 minutes at +98 C). The tested sample pretreatment protocols are summarized in Figure 1.…”

Comparison of techniques for DNA extraction and agarose gel staining of DNA fragments using samples of Cryptosporidium

“…The first protocol (Extraction Protocol I) used a commercially available Qiagen kit (QIAamp DNA Stool Mini Kit) (Geurden et al 2007;Langkjaer et al 2007;Coklin et al 2009;Das et al 2011;Adamska et al 2012), and the manufacturer's instructions were followed, with a few modifications.…”

“…Samples must contain minimal amounts of impurities to prevent inhibition of the enzymatic reactions or interference with the gel migration patterns (Romano and Brasileiro 1999;Adamska et al 2012). Therefore, the higher the purity of the extracted samples, the better the results of the nested PCR.…”

“…C. parvum is the most concerning due to its zoonotic potential (Diaz et al 2010). Bovine cryptosporidiosis is highly concerning from both an animal production and a public health perspective (Becher et al 2004;Adamska et al 2012).…”

“…Several methods for extracting Cryptosporidium DNA are described in the literature. These studies use protocols developed by researchers, commer-cial kits, or a combination of the two (Becher et al 2004;Castro-Hermida et al 2007;Feng et al 2007;Geurden et al 2007;Langkjaer et al 2007;Mendonca et al 2007;Broglia et al 2008;Coklin et al 2009;Keshavarz et al 2009;Coklin et al 2010;Diaz et al 2010;Fayer et al 2010a,b;Das et al 2011;Dixon et al 2011;Meireles et al 2011;Nazemalhosseini-Mojarad et al 2011;Adamska et al 2012).…”