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KMID : 0880220100480050586
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Journal of Microbiology
2010 Volume.48 No. 5 p.586 ~ p.593
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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
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Lee Cheong-Hoon
Cheong Soo-ryun
Lee Hee-Jung
Kwon Mi-ye
Kang Il-Nam
Oh Eun-Gyoung
Yu Hong-Sik
Shin Soon-Bum
Kim Sang-Jong
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Abstract
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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.
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KEYWORD
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norovirus, oyster, nested RT-PCR, primer sensitivity and specificity, RNA extraction, efficiency
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