Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR
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...
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| Format: | Article |
| Language: | English |
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Czech Academy of Agricultural Sciences
2012-05-01
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| Series: | Veterinární Medicína |
| Subjects: | |
| Online Access: | https://vetmed.agriculturejournals.cz/artkey/vet-201205-0002_recovery-of-cryptosporidium-from-spiked-water-and-stool-samples-measured-by-pcr-and-real-time-pcr.php |
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| author | M. Adamska A. Leonska-Duniec M. Sawczuk A. Maciejewska B. Skotarczak |
| author_facet | M. Adamska A. Leonska-Duniec M. Sawczuk A. Maciejewska B. Skotarczak |
| author_sort | M. Adamska |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T08:00:35Z |
| format | Article |
| id | doaj.art-41fcba487e04429da59016d15697bb32 |
| institution | Directory Open Access Journal |
| issn | 0375-8427 1805-9392 |
| language | English |
| last_indexed | 2024-04-10T08:00:35Z |
| publishDate | 2012-05-01 |
| publisher | Czech Academy of Agricultural Sciences |
| record_format | Article |
| series | Veterinární Medicína |
| spelling | doaj.art-41fcba487e04429da59016d15697bb322023-02-23T03:50:02ZengCzech Academy of Agricultural SciencesVeterinární Medicína0375-84271805-93922012-05-0157522423210.17221/5952-VETMEDvet-201205-0002Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCRM. Adamska0A. Leonska-Duniec1M. Sawczuk2A. Maciejewska3B. Skotarczak4Department of Genetics, University of Szczecin, PolandDepartment of Genetics, University of Szczecin, PolandDepartment of Genetics, University of Szczecin, PolandDepartment of Genetics, University of Szczecin, PolandDepartment of Genetics, University of Szczecin, PolandCryptosporidium 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.https://vetmed.agriculturejournals.cz/artkey/vet-201205-0002_recovery-of-cryptosporidium-from-spiked-water-and-stool-samples-measured-by-pcr-and-real-time-pcr.phpcryptosporidium parvum oocystsspiked water and stoolwater filtrationstool concentrationnested pcrtaqman nested real time pcrbovine serum albumin |
| spellingShingle | M. Adamska A. Leonska-Duniec M. Sawczuk A. Maciejewska B. Skotarczak Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR Veterinární Medicína cryptosporidium parvum oocysts spiked water and stool water filtration stool concentration nested pcr taqman nested real time pcr bovine serum albumin |
| title | Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR |
| title_full | Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR |
| title_fullStr | Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR |
| title_full_unstemmed | Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR |
| title_short | Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR |
| title_sort | recovery of cryptosporidium from spiked water and stool samples measured by pcr and real time pcr |
| topic | cryptosporidium parvum oocysts spiked water and stool water filtration stool concentration nested pcr taqman nested real time pcr bovine serum albumin |
| url | https://vetmed.agriculturejournals.cz/artkey/vet-201205-0002_recovery-of-cryptosporidium-from-spiked-water-and-stool-samples-measured-by-pcr-and-real-time-pcr.php |
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