Open AccessSensitive Detection of RNA Viruses Associated with Gastroenteritis by a Hanging-Drop Single-Tube Nested Reverse Transcription-PCR Method
Author(s) -
Rodney Μ. Ratcliff,
James Christopher Doherty,
Geoffrey D. Higgins
Publication year - 2002
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.40.11.4091-4099.2002
Subject(s) - astrovirus , virology , reverse transcriptase , rotavirus , biology , outbreak , norwalk virus , virus , reverse transcription polymerase chain reaction , complementary dna , primer (cosmetics) , nested polymerase chain reaction , polymerase chain reaction , norovirus , genetics , gene , chemistry , messenger rna , organic chemistry
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).