Rapid detection of Hepatitis E virus RNA by reverse transcription-polymerase chain reaction using universal oligonucleotide primers.
A rapid reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of Hepatitis E virus (HEV) RNA in serum is described. Total nucleic acids are extracted from a small volume of human serum and reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in nested PCR employing degenerate HEV consensus primers. These primers are designed to sequences conserved between the Burma, Mexico, and US HEV strains, generating amplicons within each of the three open reading frames. Reactions are analyzed by agarose gel electrophoresis and samples showing an ethidium bromide stained band of the appropriate size in the first and second amplification, or in the second amplification only, are designated as positive. This protocol allows for the rapid and sensitive detection of HEV infection in human serum.
Erker
J C
JC
Virus Discovery Group, Experimental Biology Research, Abbott Laboratories, North Chicago, IL 60064-6269, USA.
Desai
S M
SM
Mushahwar
I K
IK
D017931:DNA Primers / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D016752:Hepatitis E virus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000097:blood* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
D017931:DNA Primers; D012367:RNA, Viral