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

C.J. LOWERY^*,, J.E. MOORE, B.C. MILLAR, D.P. BURKE^*, K.A. J. McCORRY, E. CROTHERS and J.S. G. DOOLEY^*

*Department of Applied Biological and Chemical Sciences, University of Ulster, Coleraine, County Derry BT52 1SA and Northern Ireland Public Health Laboratories, Department of Bacteriology, Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland BT9 7AB

Corresponding author: Dr J. S. G. Dooley (email: JSG. Dooley{at}ulst.ac.uk).

Received 5 Aug. 1999; revised version accepted 6 Jan. 2000.

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 10³–10⁴-fold lower in environmental water samples. The use of Chelex resin and an immunomagnetic separation procedure overcame this inhibition. This provided detection levels of 10¹–10³ 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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