Evaluation of the sensitivity and specificity of primer pairs and the efficiency of RNA extraction procedures to improve noroviral detection from oysters by nested reverse transcription-polymerase chain reaction.
Noroviruses (NoV) are the key cause of acute epidemic gastroenteritis, and oysters harvested from NoV-polluted sea areas are considered as the significant vectors of viral transmission. To improve NoV detection from oyster using nested reverse transcription-polymerase chain reaction (RT-PCR), we evaluated the sensitivity and specificity of previously published primer pairs and the efficiency of different RNA extraction procedures. Among the primer pairs used for RT-PCR, the sensitivity of GIF1/GIR1-GIF2/GIR1 and GIIF1/GIIR1-GIIF2/GIIR1 was higher than that of other primer pairs used in nested RT-PCR for the detection of NoV genogroup I (NoV GI) and NoV GII from both NoV-positive stool suspension and NoV-seeded oyster concentrates, respectively; the resulting products showed neither unspecific bands in the positive samples nor false-positive bands in the negative controls. The extraction of NoV RNA from oyster samples using a QIAamp® Viral RNA Mini kit with a QIAshredder™ Homogenizer pretreatment afforded more efficient recovery (mean recovery for NoV GI and GII, 6.4%) and the procedure was less time consuming (<30 min) than most other RNA extraction procedures. The results of RNA extraction procedure and primer pairs evaluated by nested RT-PCR assay in this study can be useful for monitoring NoV contamination in oysters, which is an indicator of possible public health risks.
School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
Cheonghoon
C
Lee
Sooryun
S
Cheong
Hee-Jung
HJ
Lee
Miye
M
Kwon
Ilnam
I
Kang
Eun-Gyoung
EG
Oh
Hong-Sik
HS
Yu
Soon-Bum
SB
Shin
Sang-Jong
SJ
Kim
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000821:virology; D006801:Humans; D029322:Norovirus / Q000302:isolation & purification*; D010125:Ostreidae / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D012313:RNA