Abstract:
:Current methods for detection of Cryptosporidium parvum oocysts in water are time-consuming and difficult. We have developed a reverse transcription (RT)-PCR which can detect the presence of a single viable oocyst spiked into concentrated environmental water samples. The test is based on the detection of mRNA from a C. parvum heat shock protein (hsp). The synthesis of hsp was induced by a short 45 degrees C incubation followed by oocyst lysis by a freeze-thaw process. Hsp70 mRNA, produced only from viable oocysts, was then isolated by hybridization to oligo(dT)25-coated magnetic beads. Detection was achieved by RT-PCR amplification of a 590-bp region of hsp70 mRNA specific for C. parvum. To test the method, samples of reticulated, reservoir, bore, and river water were concentrated by chemical flocculation and Percoll-sucrose gradient centrifugation and then spiked with dilutions of oocysts. In all four of the water types examined, the detection of single oocysts was possible by RT-PCR combined with Southern hybridization. RT-PCR products were not obtained from formalin-inactivated oocysts. An RNA internal positive control fragment was synthesized that was included with each reaction to guard against RT-PCR false-negative results that may be caused by the presence of inhibitory substances. However, when the magnetic beads were used to extract and concentrate mRNA, no inhibition was observed. The technique is versatile, straightforward, and rapid (1 day) and provides a sensitive and economic means of screening concentrated water samples for the presence of C. parvum.
journal_name
Appl Environ Microbioljournal_title
Applied and environmental microbiologyauthors
Stinear T,Matusan A,Hines K,Sandery Mdoi
10.1128/AEM.62.9.3385-3390.1996subject
Has Abstractpub_date
1996-09-01 00:00:00pages
3385-90issue
9eissn
0099-2240issn
1098-5336journal_volume
62pub_type
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doi:10.1128/AEM.65.4.1696-1702.1999
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journal_title:Applied and environmental microbiology
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doi:10.1128/AEM.41.6.1374-1377.1981
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journal_title:Applied and environmental microbiology
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doi:10.1128/AEM.41.3.652-656.1981
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pub_type: 杂志文章
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.64.9.3525-3529.1998
更新日期:1998-09-01 00:00:00
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doi:10.1128/AEM.49.6.1379-1384.1985
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pub_type: 杂志文章
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.34.4.347-350.1977
更新日期:1977-10-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.54.6.1399-1404.1988
更新日期:1988-06-01 00:00:00
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pub_type: 杂志文章
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.53.12.2714-2724.1987
更新日期:1987-12-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.43.3.731-733.1982
更新日期:1982-03-01 00:00:00
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