Development of a sensitive detection system for Cryptosporidium in environmental samples
The identification of Cryptosporidium species and genotypes is necessary to determine sources of infection in outbreaks and the risk factors associated with their transmission. Few studies have applied isolation methods to field samples because of difficulties with detection of oocysts in environmen...
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| Veröffentlicht in: | Veterinary parasitology 2006-03, Vol.136 (3), p.201-213 |
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| creator | Ramirez, Norma E. Sreevatsan, Srinand |
| description | The identification of
Cryptosporidium species and genotypes is necessary to determine sources of infection in outbreaks and the risk factors associated with their transmission. Few studies have applied isolation methods to field samples because of difficulties with detection of oocysts in environmental samples, particularly in soil and manure. The objective of this study was to develop an easy to use method which can be applied to field samples to rapidly detect the presence of
Cryptosporidium parasites and identify their species. The assay included an oocyst recovery method combined with spin column DNA extraction, followed by PCR-hybridization for detection and a real-time PCR-melting curve analysis for species assignment. An internal positive control (IPC) was developed to determine the presence of PCR inhibitory substances. Two oocyst recovery methods, sodium chloride and sucrose flotation techniques were compared. Two commercial DNA extraction kits were performed using feces, soil and water samples each inoculated with different concentration of
Cryptosporidium oocysts. Subsequently, methods were used to test field samples. The sucrose flotation method provided the greatest analytical sensitivity detecting as few as 10 oocysts. The PCR-hybridization detection limit was 10 oocysts for feces and soil, and less than 10 oocysts for water samples. IPC was positive for all inoculated and field samples indicating 0% PCR inhibition.
Cryptosporidium species DNA samples were detected with the real-time PCR and were differentiated by the melting curve analysis. The results of this study demonstrate the potential of the assay system for rapid detection of
Cryptosporidium parasites in environmental samples. |
| doi_str_mv | 10.1016/j.vetpar.2005.11.023 |
| format | Article |
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Cryptosporidium species and genotypes is necessary to determine sources of infection in outbreaks and the risk factors associated with their transmission. Few studies have applied isolation methods to field samples because of difficulties with detection of oocysts in environmental samples, particularly in soil and manure. The objective of this study was to develop an easy to use method which can be applied to field samples to rapidly detect the presence of
Cryptosporidium parasites and identify their species. The assay included an oocyst recovery method combined with spin column DNA extraction, followed by PCR-hybridization for detection and a real-time PCR-melting curve analysis for species assignment. An internal positive control (IPC) was developed to determine the presence of PCR inhibitory substances. Two oocyst recovery methods, sodium chloride and sucrose flotation techniques were compared. Two commercial DNA extraction kits were performed using feces, soil and water samples each inoculated with different concentration of
Cryptosporidium oocysts. Subsequently, methods were used to test field samples. The sucrose flotation method provided the greatest analytical sensitivity detecting as few as 10 oocysts. The PCR-hybridization detection limit was 10 oocysts for feces and soil, and less than 10 oocysts for water samples. IPC was positive for all inoculated and field samples indicating 0% PCR inhibition.
Cryptosporidium species DNA samples were detected with the real-time PCR and were differentiated by the melting curve analysis. The results of this study demonstrate the potential of the assay system for rapid detection of
Cryptosporidium parasites in environmental samples.</description><identifier>ISSN: 0304-4017</identifier><identifier>EISSN: 1873-2550</identifier><identifier>DOI: 10.1016/j.vetpar.2005.11.023</identifier><identifier>PMID: 16387443</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>Animals ; Area Under Curve ; Base Sequence ; contamination ; Cryptosporidium ; Cryptosporidium - isolation & purification ; Detection ; Disease Reservoirs - veterinary ; DNA ; DNA extraction ; DNA Primers ; DNA, Protozoan - analysis ; Environment ; environmental sampling ; extraction ; feces ; Feces - parasitology ; Hybridization ; Internal positive control ; Manure - parasitology ; methodology ; oocysts ; Oocysts - isolation & purification ; pathogen identification ; polymerase chain reaction ; Polymerase Chain Reaction - methods ; Polymerase Chain Reaction - veterinary ; Real-time PCR ; sampling ; Sensitivity and Specificity ; Soil - parasitology ; soil sampling ; Temperature ; water ; Water - parasitology</subject><ispartof>Veterinary parasitology, 2006-03, Vol.136 (3), p.201-213</ispartof><rights>2005 Elsevier B.V.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c415t-63f7d728081aab0ee090ddece29196c162680b0ef70704e99a32383cd93ba29b3</citedby><cites>FETCH-LOGICAL-c415t-63f7d728081aab0ee090ddece29196c162680b0ef70704e99a32383cd93ba29b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0304401705005637$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16387443$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ramirez, Norma E.</creatorcontrib><creatorcontrib>Sreevatsan, Srinand</creatorcontrib><title>Development of a sensitive detection system for Cryptosporidium in environmental samples</title><title>Veterinary parasitology</title><addtitle>Vet Parasitol</addtitle><description>The identification of
Cryptosporidium species and genotypes is necessary to determine sources of infection in outbreaks and the risk factors associated with their transmission. Few studies have applied isolation methods to field samples because of difficulties with detection of oocysts in environmental samples, particularly in soil and manure. The objective of this study was to develop an easy to use method which can be applied to field samples to rapidly detect the presence of
Cryptosporidium parasites and identify their species. The assay included an oocyst recovery method combined with spin column DNA extraction, followed by PCR-hybridization for detection and a real-time PCR-melting curve analysis for species assignment. An internal positive control (IPC) was developed to determine the presence of PCR inhibitory substances. Two oocyst recovery methods, sodium chloride and sucrose flotation techniques were compared. Two commercial DNA extraction kits were performed using feces, soil and water samples each inoculated with different concentration of
Cryptosporidium oocysts. Subsequently, methods were used to test field samples. The sucrose flotation method provided the greatest analytical sensitivity detecting as few as 10 oocysts. The PCR-hybridization detection limit was 10 oocysts for feces and soil, and less than 10 oocysts for water samples. IPC was positive for all inoculated and field samples indicating 0% PCR inhibition.
Cryptosporidium species DNA samples were detected with the real-time PCR and were differentiated by the melting curve analysis. The results of this study demonstrate the potential of the assay system for rapid detection of
Cryptosporidium parasites in environmental samples.</description><subject>Animals</subject><subject>Area Under Curve</subject><subject>Base Sequence</subject><subject>contamination</subject><subject>Cryptosporidium</subject><subject>Cryptosporidium - isolation & purification</subject><subject>Detection</subject><subject>Disease Reservoirs - veterinary</subject><subject>DNA</subject><subject>DNA extraction</subject><subject>DNA Primers</subject><subject>DNA, Protozoan - analysis</subject><subject>Environment</subject><subject>environmental sampling</subject><subject>extraction</subject><subject>feces</subject><subject>Feces - parasitology</subject><subject>Hybridization</subject><subject>Internal positive control</subject><subject>Manure - parasitology</subject><subject>methodology</subject><subject>oocysts</subject><subject>Oocysts - isolation & purification</subject><subject>pathogen identification</subject><subject>polymerase chain reaction</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Polymerase Chain Reaction - veterinary</subject><subject>Real-time PCR</subject><subject>sampling</subject><subject>Sensitivity and Specificity</subject><subject>Soil - parasitology</subject><subject>soil sampling</subject><subject>Temperature</subject><subject>water</subject><subject>Water - parasitology</subject><issn>0304-4017</issn><issn>1873-2550</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkVGL1DAQx4Mo3t7pNxDNk2_tzSTdpH0RZNXz4MCHuwPfQraZSpa2qUm2sN_eLl3wTZ8Ght__z_Abxt4hlAiobg_lTHmysRQA2xKxBCFfsA3WWhZiu4WXbAMSqqIC1FfsOqUDAFSg9Gt2hUrWuqrkhv38QjP1YRpozDx03PJEY_LZz8QdZWqzDyNPp5Rp4F2IfBdPUw5pCtE7fxy4HzmNs49hPFfYnic7TD2lN-xVZ_tEby_zhj1_-_q0-148_Li7331-KNoKt7lQstNOixpqtHYPRNCAc9SSaLBRLSqhalj2nQYNFTWNlULWsnWN3FvR7OUN-7j2TjH8PlLKZvCppb63I4VjMkprgajgvyBqULWumwWsVrCNIaVInZmiH2w8GQRzVm8OZlVvzuoNolnUL7H3l_7jfiD3N3RxvQAfVqCzwdhf0Sfz_CgAJSBo2WixEJ9WghZhs6doUutpbMn5uHzCuOD_fcMfl-qhSw</recordid><startdate>20060331</startdate><enddate>20060331</enddate><creator>Ramirez, Norma E.</creator><creator>Sreevatsan, Srinand</creator><general>Elsevier B.V</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7UA</scope><scope>C1K</scope><scope>F1W</scope><scope>H97</scope><scope>L.G</scope><scope>M7N</scope><scope>7X8</scope></search><sort><creationdate>20060331</creationdate><title>Development of a sensitive detection system for Cryptosporidium in environmental samples</title><author>Ramirez, Norma E. ; Sreevatsan, Srinand</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c415t-63f7d728081aab0ee090ddece29196c162680b0ef70704e99a32383cd93ba29b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Animals</topic><topic>Area Under Curve</topic><topic>Base Sequence</topic><topic>contamination</topic><topic>Cryptosporidium</topic><topic>Cryptosporidium - isolation & purification</topic><topic>Detection</topic><topic>Disease Reservoirs - veterinary</topic><topic>DNA</topic><topic>DNA extraction</topic><topic>DNA Primers</topic><topic>DNA, Protozoan - analysis</topic><topic>Environment</topic><topic>environmental sampling</topic><topic>extraction</topic><topic>feces</topic><topic>Feces - parasitology</topic><topic>Hybridization</topic><topic>Internal positive control</topic><topic>Manure - parasitology</topic><topic>methodology</topic><topic>oocysts</topic><topic>Oocysts - isolation & purification</topic><topic>pathogen identification</topic><topic>polymerase chain reaction</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Polymerase Chain Reaction - veterinary</topic><topic>Real-time PCR</topic><topic>sampling</topic><topic>Sensitivity and Specificity</topic><topic>Soil - parasitology</topic><topic>soil sampling</topic><topic>Temperature</topic><topic>water</topic><topic>Water - parasitology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ramirez, Norma E.</creatorcontrib><creatorcontrib>Sreevatsan, Srinand</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Water Resources Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 3: Aquatic Pollution & Environmental Quality</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>MEDLINE - Academic</collection><jtitle>Veterinary parasitology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ramirez, Norma E.</au><au>Sreevatsan, Srinand</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of a sensitive detection system for Cryptosporidium in environmental samples</atitle><jtitle>Veterinary parasitology</jtitle><addtitle>Vet Parasitol</addtitle><date>2006-03-31</date><risdate>2006</risdate><volume>136</volume><issue>3</issue><spage>201</spage><epage>213</epage><pages>201-213</pages><issn>0304-4017</issn><eissn>1873-2550</eissn><abstract>The identification of
Cryptosporidium species and genotypes is necessary to determine sources of infection in outbreaks and the risk factors associated with their transmission. Few studies have applied isolation methods to field samples because of difficulties with detection of oocysts in environmental samples, particularly in soil and manure. The objective of this study was to develop an easy to use method which can be applied to field samples to rapidly detect the presence of
Cryptosporidium parasites and identify their species. The assay included an oocyst recovery method combined with spin column DNA extraction, followed by PCR-hybridization for detection and a real-time PCR-melting curve analysis for species assignment. An internal positive control (IPC) was developed to determine the presence of PCR inhibitory substances. Two oocyst recovery methods, sodium chloride and sucrose flotation techniques were compared. Two commercial DNA extraction kits were performed using feces, soil and water samples each inoculated with different concentration of
Cryptosporidium oocysts. Subsequently, methods were used to test field samples. The sucrose flotation method provided the greatest analytical sensitivity detecting as few as 10 oocysts. The PCR-hybridization detection limit was 10 oocysts for feces and soil, and less than 10 oocysts for water samples. IPC was positive for all inoculated and field samples indicating 0% PCR inhibition.
Cryptosporidium species DNA samples were detected with the real-time PCR and were differentiated by the melting curve analysis. The results of this study demonstrate the potential of the assay system for rapid detection of
Cryptosporidium parasites in environmental samples.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>16387443</pmid><doi>10.1016/j.vetpar.2005.11.023</doi><tpages>13</tpages></addata></record> |
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| subjects | Animals Area Under Curve Base Sequence contamination Cryptosporidium Cryptosporidium - isolation & purification Detection Disease Reservoirs - veterinary DNA DNA extraction DNA Primers DNA, Protozoan - analysis Environment environmental sampling extraction feces Feces - parasitology Hybridization Internal positive control Manure - parasitology methodology oocysts Oocysts - isolation & purification pathogen identification polymerase chain reaction Polymerase Chain Reaction - methods Polymerase Chain Reaction - veterinary Real-time PCR sampling Sensitivity and Specificity Soil - parasitology soil sampling Temperature water Water - parasitology |
| title | Development of a sensitive detection system for Cryptosporidium in environmental samples |
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