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Appl Environ Microbiol, May 1998, p. 1743-1749, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Sensitive and Rapid Detection of Viable
Giardia Cysts and Cryptosporidium parvum Oocysts
in Large-Volume Water Samples with Wound Fiberglass Cartridge
Filters and Reverse Transcription-PCR
Christine
Kaucner* and
Timothy
Stinear
WATER ECOscience Pty. Ltd., Mount Waverley,
Victoria 3149, Australia
Received 30 December 1997/Accepted 9 March 1998
We recently described a reverse transcription-PCR (RT-PCR) for
detecting low numbers of viable Cryptosporidium parvum
oocysts spiked into clarified environmental water concentrates. We have now modified the assay for direct analysis of primary sample
concentrates with simultaneous detection of viable C. parvum oocysts, Giardia cysts, and a novel type of
internal positive control (IPC). The IPC was designed to assess both
efficiency of mRNA isolation and potential RT-PCR inhibition.
Sensitivity testing showed that low numbers of organisms, in the range
of a single viable cyst and oocyst, could be detected when spiked into
100-µl packed pellet volumes of concentrates from creek and river
water samples. The RT-PCR was compared with an immunofluorescence (IF)
assay by analyzing 29 nonspiked environmental water samples. Sample
volumes of 20 to 1,500 liters were concentrated with a wound fiberglass
cartridge filter. Frequency of detection for viable Giardia
cysts increased from 24% by IF microscopy to 69% by RT-PCR. Viable
C. parvum oocysts were detected only once by RT-PCR (3%)
in contrast to detection of viable Cryptosporidium spp. in
four samples by IF microscopy (14%), suggesting that
Cryptosporidium species other than C. parvum were present in the water. This combination of the large-volume sampling method with RT-PCR represents a significant advance in terms
of protozoan pathogen monitoring and in the wider application of PCR
technology to this field of microbiology.
*
Corresponding author. Mailing address: WATER ECOscience
Pty. Ltd., 68 Ricketts Rd., Mount Waverley, Victoria 3149, Australia. Phone: 61 3 9550 1031. Fax: 61 3 9543 7185. E-mail:
ckaucner{at}wes.com.au.
Appl Environ Microbiol, May 1998, p. 1743-1749, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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