Detection and speciation of Cryptosporidium spp. in environmental water samples by immunomagnetic separation, PCR and endonuclease restriction

Colm Lowery, JE Moore, BC Millar, DP Burke, KAJ McCorry, E Crothers, James Dooley

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

Current methods for the detection of Cryptosporidium oocysts in water samples are both time-consuming and subject to variation in sensitivity. A genus-specific PCR assay was designed for the specific amplification of a 552-bp region of the 18S rRNA gene. Post-amplification endonuclease restriction generated unique digest patterns that enabled differentiation between the three species, C, muris, C, baileyi and C. parvum, the major human pathogen. Theoretical restriction profiles for other Cryptosporidium species were also predicted, The assay routinely detected 10 oocysts in 10-ml purified oocyst preparations, but sensitivity was found to be 10(3)-10(4)-fold lower in environmental water samples. The use of Chelex resin and an immunomagnetic separation procedure overcame this inhibition. This provided detection levels of 10(1)-10(3) oocysts, depending on water turbidity, Rapid and sensitive pathogen detection methods are essential for the water industry. The results of this study demonstrate that PCR has the potential to improve current detection capabilities greatly by differentiating the major human pathogens from non-pathogenic species. This will greatly facilitate a closer examination of the epidemiology of this important pathogen.
LanguageEnglish
Pages779-785
JournalJournal of Medical Microbiology
Volume49
Issue number9
Publication statusPublished - Sep 2000

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DNA Restriction Enzymes
Pathogens
Water
Amplification
Assays
Epidemiology
Turbidity
Resins
Genes
Industry

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title = "Detection and speciation of Cryptosporidium spp. in environmental water samples by immunomagnetic separation, PCR and endonuclease restriction",
abstract = "Current methods for the detection of Cryptosporidium oocysts in water samples are both time-consuming and subject to variation in sensitivity. A genus-specific PCR assay was designed for the specific amplification of a 552-bp region of the 18S rRNA gene. Post-amplification endonuclease restriction generated unique digest patterns that enabled differentiation between the three species, C, muris, C, baileyi and C. parvum, the major human pathogen. Theoretical restriction profiles for other Cryptosporidium species were also predicted, The assay routinely detected 10 oocysts in 10-ml purified oocyst preparations, but sensitivity was found to be 10(3)-10(4)-fold lower in environmental water samples. The use of Chelex resin and an immunomagnetic separation procedure overcame this inhibition. This provided detection levels of 10(1)-10(3) oocysts, depending on water turbidity, Rapid and sensitive pathogen detection methods are essential for the water industry. The results of this study demonstrate that PCR has the potential to improve current detection capabilities greatly by differentiating the major human pathogens from non-pathogenic species. This will greatly facilitate a closer examination of the epidemiology of this important pathogen.",
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Detection and speciation of Cryptosporidium spp. in environmental water samples by immunomagnetic separation, PCR and endonuclease restriction. / Lowery, Colm; Moore, JE; Millar, BC; Burke, DP; McCorry, KAJ; Crothers, E; Dooley, James.

In: Journal of Medical Microbiology, Vol. 49, No. 9, 09.2000, p. 779-785.

Research output: Contribution to journalArticle

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AU - Moore, JE

AU - Millar, BC

AU - Burke, DP

AU - McCorry, KAJ

AU - Crothers, E

AU - Dooley, James

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AB - Current methods for the detection of Cryptosporidium oocysts in water samples are both time-consuming and subject to variation in sensitivity. A genus-specific PCR assay was designed for the specific amplification of a 552-bp region of the 18S rRNA gene. Post-amplification endonuclease restriction generated unique digest patterns that enabled differentiation between the three species, C, muris, C, baileyi and C. parvum, the major human pathogen. Theoretical restriction profiles for other Cryptosporidium species were also predicted, The assay routinely detected 10 oocysts in 10-ml purified oocyst preparations, but sensitivity was found to be 10(3)-10(4)-fold lower in environmental water samples. The use of Chelex resin and an immunomagnetic separation procedure overcame this inhibition. This provided detection levels of 10(1)-10(3) oocysts, depending on water turbidity, Rapid and sensitive pathogen detection methods are essential for the water industry. The results of this study demonstrate that PCR has the potential to improve current detection capabilities greatly by differentiating the major human pathogens from non-pathogenic species. This will greatly facilitate a closer examination of the epidemiology of this important pathogen.

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