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Journal of Clinical Microbiology, November 2002, p. 4091-4099, Vol. 40, No. 11
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.11.4091-4099.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Sensitive Detection of RNA Viruses Associated with Gastroenteritis by a Hanging-Drop Single-Tube Nested Reverse Transcription-PCR Method

Rodney Mark Ratcliff,* James Christopher Doherty, and Geoffrey David Higgins

Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Adelaide, South Australia 5000, Australia

Received 30 November 2001/ Returned for modification 18 February 2002/ Accepted 2 July 2002

The detection of the human RNA viruses, calicivirus and astrovirus, requires high sensitivity and broad reactivity. A novel single-tube nested reverse transcription-PCR (RT-PCR) method is described here, in which all of the required reagents are included in the one tube; however, those required for the nested amplification are separated in a "hanging drop" in the cap to be introduced by centrifugation after the RT and first-round cDNA amplification steps. Broad reactivity was obtained by using primer cocktails covering the published sequence variation in the primer targets. The method was evaluated with clinical fecal samples from outbreak and sporadic cases. Norwalk-like virus types 1 and 2 and rotavirus were the causal agents in 10 of 12 outbreaks. A viral agent was detected in 44% of 197 samples from sporadic infections in patients presenting to community health centers and a children's hospital. Interestingly, whereas rotavirus was more common than astrovirus in patients presenting to the hospital (33 and 7.6%, respectively), the reverse was true for patients presenting to community health centers (4.2 and 34%, respectively).

* Corresponding author. Mailing address: Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, P.O. Box 14, Rundle Mall, Adelaide, South Australia 5000, Australia. Phone: 61-8-82223321. Fax: 61-8-82223543. E-mail: rod.ratcliff{at}imvs.sa.gov.au.

Journal of Clinical Microbiology, November 2002, p. 4091-4099, Vol. 40, No. 11
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.11.4091-4099.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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