Increased detection of rotavirus using a real time reverse transcription‐polymerase chain reaction (RT‐PCR) assay in stool specimens from children with diarrhea

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Abstract
Six‐hundred and twenty‐six stool specimens collected from children with diarrhea over a 12‐month period were tested for rotavirus using a real time reverse transcription‐polymerase chain reaction (RT‐PCR) assay, a conventional nested PCR assay and by electron microscopy (EM). A fragment of 87 bp in a highly‐conserved region of non‐structural protein 3 (NSP3) in rotavirus genome was amplified by a single‐step RT‐PCR protocol in a closed‐tube system. Rotavirus was detected in 123 samples (20%) with the real time RT‐PCR assay, 113 samples (18%) with the nested‐PCR assay, and 79 samples (13%) with EM. Using serial diluted nucleic acid extract, we compared the sensitivity of real time RT‐PCR with conventional RT‐PCR and conventional nested PCR assays. Real time RT‐PCR was two to four logs more sensitive than the conventional assays. The reaction time required for the RT‐PCR assay is about half the time required for the conventional nested‐PCR. The real time RT‐PCR assay is both simple and rapid with advantages including enhanced sensitivity and a lower risk for cross‐contamination making it a useful tool for the detection of rotavirus in various situations including sporadic gastroenteritis, outbreaks, and environmental investigations. G1 was the predominant type (89%), followed by G2 (10%), and G4 (1%). No rotavirus of G3, G8, and G9 types were found. The peak season for rotavirus infection was January to May in northern Alberta. J. Med. Virol. 72:496–501, 2004.