Sensitive detection of RNA viruses associated with gastroenteritis by a hanging-drop single-tube nested reverse transcription-PCR method.

Abstract:

: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).

journal_name

J Clin Microbiol

authors

Ratcliff RM,Doherty JC,Higgins GD

doi

10.1128/jcm.40.11.4091-4099.2002

subject

Has Abstract

pub_date

2002-11-01 00:00:00

pages

4091-9

issue

11

eissn

0095-1137

issn

1098-660X

journal_volume

40

pub_type

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