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Applied and Environmental Microbiology, September 2006, p. 5857-5863, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00113-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Rapid, Sensitive, and Discriminating Identification of Naegleria spp. by Real-Time PCR and Melting-Curve Analysis

Bret S. Robinson,^* Paul T. Monis, and Phillip J. Dobson

Australian Water Quality Centre, South Australia Water Corporation, Private Mail Bag 3, Salisbury, South Australia 5108, Australia

Received 16 January 2006/ Accepted 27 June 2006

The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.

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* Corresponding author. Mailing address: Australian Water Quality Centre, Private Mail Bag 3, Salisbury, SA 5108, Australia. Phone: 61 8 82590341. Fax: 61 8 82590228. E-mail: bret.robinson{at}sawater.com.au.

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Applied and Environmental Microbiology, September 2006, p. 5857-5863, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00113-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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