Detection of noroviruses in fecal specimens by direct RT-PCR without RNA purification.
Noroviruses are important human pathogens which cause epidemic acute viral gastroenteritis. Current techniques used for detection of noroviruses in fecal specimens involve multi-step viral RNA extraction and purification followed by reverse transcriptase-polymerase chain reaction (RT-PCR). This study demonstrates a method for easy detection of norovirus in fecal specimens, involving one-step RNA release and direct use of the released RNA for RT-PCR (direct RT-PCR). For one-step RNA release, a simple method was adopted based on addition of the sample treatment reagent from a commercialized Norovirus GI and GII RNA Detection Kit to suspended fecal specimens, followed by a brief heat treatment. The released RNA was then added directly to the RT mixture from the same kit. After reverse transcription and PCR, the product was detected by agarose gel electrophoresis. Direct RT-PCR was evaluated with 275 fecal specimens comprising 230 norovirus-positive and 45 norovirus-negative samples as assessed by real-time RT-PCR, considered to be the "gold standard" for norovirus detection. Direct RT-PCR was sufficiently specific and sensitive for norovirus detection, and eliminated the RNA extraction and purification step. Use of this method should facilitate detection of norovirus in fecal specimens and provide valuable information regarding the incidence of the virus. In addition, this method should be applicable for other RNA viruses.
Nishimura
Naoyuki
N
Analytical & Measuring Instruments Division, Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto 604-8511, Japan. nisimura@shimadzu.co.jp
Nakayama
Hiroyuki
H
Yoshizumi
Shima
S
Miyoshi
Masahiro
M
Tonoike
Hiroshi
H
Shirasaki
Yoshinari
Y
Kojima
Kouichi
K
Ishida
Setsuko
S
D017250:Caliciviridae Infections / Q000175:diagnosis*; D004587:Electrophoresis, Agar Gel; D005243:Feces / Q000821:virology*; D005759:Gastroenteritis / Q000821:virology*; D006801:Humans; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D012367:RNA, Viral