Genotyping of single Cryptosporidium oocysts in sewage by semi-nested PCR and direct sequencing

Hashimoto, Atsushi; Sugimoto, Hiroyuki; Morita, Shigemitsu; Hirata, Tsuyoshi

doi:10.1016/j.watres.2006.04.038

Cited by 27 publications

(15 citation statements)

References 30 publications

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“…Surprisingly, urban wastewater discharge was not found to be a significant contributor to Cryptosporidium oocyst contamination in river water in this study. Although two watershed sites (the Great Seneca Creek and Monocacy River sites) are downstream of discharges of treated urban wastewater and the two downstream treatment intake sites are under the cumulative influence of all the environmental factors considered in this study, the most common Cryptosporidium genotypes in raw urban wastewater, C. hominis and C. parvum (11,13,53,57,62,63), were never found in river water samples in this study. This finding differs from the results of a previous study conducted in neighboring areas.…”

Section: Discussionmentioning

confidence: 71%

Cryptosporidium Source Tracking in the Potomac River Watershed

Yang

Chen²,

Villegas

et al. 2008

Appl Environ Microbiol

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To better characterize Cryptosporidium in the Potomac River watershed, a PCR-based genotyping tool was used to analyze 64 base flow and 28 storm flow samples from five sites in the watershed. These sites included two water treatment plant intakes, as well as three upstream sites, each associated with a different type of land use. The uses, including urban wastewater, agricultural (cattle) wastewater, and wildlife, posed different risks in terms of the potential contribution of Cryptosporidium oocysts to the source water. Cryptosporidium was detected in 27 base flow water samples and 23 storm flow water samples. The most frequently detected species was C. andersoni (detected in 41 samples), while 14 other species or genotypes, almost all wildlife associated, were occasionally detected. The two common human-pathogenic species, C. hominis and C. parvum, were not detected. Although C. andersoni was common at all four sites influenced by agriculture, it was largely absent at the urban wastewater site. There were very few positive samples as determined by Environmental Protection Agency method 1623 at any site; only 8 of 90 samples analyzed (9%) were positive for Cryptosporidium as determined by microscopy. The genotyping results suggest that many of the Cryptosporidium oocysts in the water treatment plant source waters were from old calves and adult cattle and might not pose a significant risk to human health.

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Section: Discussionmentioning

confidence: 71%

Cryptosporidium Source Tracking in the Potomac River Watershed

Yang

Chen²,

Villegas

et al. 2008

Appl Environ Microbiol

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“…Accordingly, DNA extraction from formalin-fixed samples is affected by factors such as incomplete cell lysis, crosslinkage with the protein, coextraction of PCR inhibitors and degradation of DNA (Jara et al 2008;Santos et al 2008). The failure of sequence amplification is often associated with low quantity and quality of DNA extracted from this kind of samples, and the sensitivity of PCR drops with the decrease in DNA template (Hashimoto et al 2006). Nested and semi-nested PCR have been proven powerful to improve the specificity and sensitivity of PCR amplifications (Cowley et al 2004;Hashimoto et al 2006).…”

Section: Introductionmentioning

confidence: 99%

Exploiting formalin‐preserved fish specimens for resources of DNA barcoding

Zhang

2010

Molecular Ecology Resources

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With the development of the DNA barcoding project, a large number of specimens are required to establish the library of reference barcode. Formalin-fixed samples from museums provide a potential resource for it. However, recovery of DNA and amplification of the target gene from formalin-fixed samples are challenging. In this study, a hot alkali pre-treatment accompanied by the use of cetyltrimethylammonium bromide (CTAB) method was employed for DNA recovery from formalin-preserved samples, with the purpose of pursuing the optimal condition for high quantity and quality of DNA and minimizing PCR inhibition. Meanwhile, a semi-nested PCR-based method was developed to enhance the efficacy of amplification. This advanced protocol was demonstrated to be reliable and effective. Even for 23-year-old samples, genomic DNA could be extracted, and COI gene was correctly sequenced.

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“…Moreover, limitations of RFLP resulted in some isolates identified as mixtures or atypical genotypes (14.5%). Therefore, to complete the Cryptosporidium identification and characterisation sometimes it is necessary to apply other molecular techniques, like cloning and sequencing (Gòmez-Couso et al 2004, 2005Hashimoto et al 2006;Trotz-Williams et al 2006). Indeed, molecular tools improved our PCR-RFLP results.…”

Section: Resultsmentioning

confidence: 99%

“…PCR-based genotyping techniques may target several housekeeping or structural genes, such as the oocyst wall protein (Cryptosporidium parvum oocyst wall protein (COWP); Sulaiman et al 1999). Nested PCR procedures were shown to increase considerably the amplification sensitivity of Cryptosporidium DNA extracted from water (Sturbaum et al 2001), sewage (Hashimoto et al 2006) and faecal samples (Pedraza-Dìaz et al 2001;Amar et al 2004;Trotz-Williams et al 2006).…”

Section: Introductionmentioning

confidence: 99%

Detection of Cryptosporidium spp. from human faeces by PCR-RFLP, cloning and sequencing

et al. 2008

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In this study, we compared and validated a nested ABC polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay to amplify Cryptosporidium parvum oocyst wall protein (COWP) gene fragment with a previous nested PCR-RFLP method. When RFLP of COWP gene did not provide clear results, we further analysed samples by cloning and sequencing for species and genotype identification. ABC-PCR was performed on DNA extracted from human faecal samples collected in England where Cryptosporidium was previously detected by conventional methods. COWP gene amplification was successful for all specimens; RFLP analyses using both RsaI and AluI enzymes recognised 54/55 restriction profiles. Among these, 40 (72.7%) characteristic patterns were generated for C. parvum and six (10.9%) for Cryptosporidium hominis; four (7.3%) showed species mixtures, while the remaining four samples (7.3%) showed atypical genotypes. Cloning and sequencing of mixtures and atypical genotypes identified one C. parvum, one C. hominis and three Cryptosporidium meleagridis. Indeed, molecular tools improved PCR-RFLP results. In conclusion, this study showed that ABC-PCR-RFLP technique was quick, simple and specific. Cloning and sequencing were helpful in characterising mixtures or previously uncharacterised RFLP patterns. All these epidemiological findings are useful information for any preventive intervention to control disease diffusion.

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Genotyping of single Cryptosporidium oocysts in sewage by semi-nested PCR and direct sequencing

Cited by 27 publications

References 30 publications

Cryptosporidium Source Tracking in the Potomac River Watershed

Cryptosporidium Source Tracking in the Potomac River Watershed

Exploiting formalin‐preserved fish specimens for resources of DNA barcoding

Detection of Cryptosporidium spp. from human faeces by PCR-RFLP, cloning and sequencing

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