Molecular Characterization of
            <i>Cryptosporidium</i>
            Oocysts in Samples of Raw Surface Water and Wastewater

Xiao, Lihua; Singh, Ajmer; Limor, Josef; Graczyk, Thaddeus K.; Gradus, Steve; Lal, Altaf A.

doi:10.1128/aem.67.3.1097-1101.2001

Cited by 293 publications

(243 citation statements)

References 32 publications

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“…PCR has been considered as a sensitive method for detecting oocysts in environmental samples (Xiao et al 2001). However, nine samples were microscopically positive but negative by PCR in this study.…”

Section: Initial Precision and Recovery Of Cryptosporidium Parvum Ooccontrasting

confidence: 41%

Occurrences and genotypes of Cryptosporidium oocysts in river network of southern-eastern China

Xiao

Chen

et al. 2011

Parasitol Res

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Transportation of Cryptosporidium oocysts in river type source water is of great concern in an area where extensive human activities exist. In this study, a total of 47 samples were collected from Tongxiang, China, where drinking source water was taken from a complicated river network system, by three sampling campaigns over a rainy season in 2009, to reveal the presence, genotypes, and likely source of Cryptosporidium oocysts in river water. Immunofluorescence microscopy analyses show that 37 (78.7%) were Cryptosporidium positive, with a mean concentration of 0.51 oocysts per liter. These results suggest that the protozoa were commonly distributed in the river network type source water of Tongxiang with a relatively low concentration level. PCR analysis was used to determine the species/genotypes of Cryptosporidium, which revealed the presence of the animal related species/ genotypes including Cryptosporidium suis, Cryptosporidium fragile, and the avian III, pig II, cervine genotypes. Three of them were also detected in wastewater samples taken from neighboring animal farms, showing that farm animals rather than human might be the major pollution sources. This is the first report on simultaneous detection and genotyping of Cryptosporidium oocysts from surface water in China.

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Section: Initial Precision and Recovery Of Cryptosporidium Parvum Ooccontrasting

confidence: 41%

Occurrences and genotypes of Cryptosporidium oocysts in river network of southern-eastern China

Xiao

Chen

et al. 2011

Parasitol Res

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“…Although immunomagnetic separation (IMS) is now more commonly used and many researches have been used the method along with PCR for detection of Cryptosporidium oocysts in environmental samples (Hallier-Soulier and Guillot, 1996;Hallier-Soulier and Guillot, 2000;Rimhanen-finne, et al, 2001;Sturbaum, et al, 2002;Xiao, et al, 2001), but the procedure selectively separate the Cryptosporidium oocysts. However it would be advantageous to perform parallel detection assay for multiple pathogen on a single sample.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of QIAamp DNA mini kit for removing of inhibitors in detection of Cryptosporidium parvum oocysts in water samples by a nested-PCR assay

Nikaeen

Makimura

2007

Int. J. Environ. Sci. Technol.

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ABSTRACT:In recent years, there has been a dramatic increase in the occurrence of waterborne disease outbreaks caused by the Cryptosporidium parvum, and presence of this protozoan parasite in drinking water is a significant health problem faced by the water industry. A new strategy for detection of Cryptosporidium oocysts in water samples is PCR-based techniques. In this study a nested-PCR assay was designed for the specific amplification of a 199 bp DNA fragment of the gene encoding the heat shock protein (hsp70) of Cryptosporidium parvum oocysts. In order to prevent the inhibition of PCR amplification by substances contained in water samples, three DNA purification methods including QIAamp DNA mini kit, InstaGene Matrix, MagExtractor -Genome were compared in concentrates of tap water samples spiked with the oocysts. After it was found that the QIAamp is only efficient purification technique, the efficiency of QIAamp and immunomagnetic separation for nested-PCR assay of various water samples was compared. The results show that QIAamp provide a useful and rapid tool for removing of PCR inhibitors. It seems that QIAamp purificationnested PCR assay is a sensitive, rapid and cost effective method for detection of Cryptosporidium parvum oocysts in clean water samples with turbidity < 2 nephelometric turbidity unit (NTU).

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“…The reactions were performed in a DNA thermal cycler (Biometra, Germany and MJ Research, USA). Thermal-time profiles in the first and second PCR were the same as described by Xiao et al (2001). Negative control reaction mixtures contained sterile distilled water in place of DNA template.…”

Section: Nested Pcr Amplificationmentioning

confidence: 99%

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

Adamska¹,

Leońska-Duniec²,

Sawczuk³

et al. 2012

Vet. Med.

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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.

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Molecular Characterization of Cryptosporidium Oocysts in Samples of Raw Surface Water and Wastewater

Cited by 293 publications

References 32 publications

Occurrences and genotypes of Cryptosporidium oocysts in river network of southern-eastern China

Occurrences and genotypes of Cryptosporidium oocysts in river network of southern-eastern China

Evaluation of QIAamp DNA mini kit for removing of inhibitors in detection of Cryptosporidium parvum oocysts in water samples by a nested-PCR assay

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

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