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

Cheonghoon Lee; Sooryun Cheong; Hee-Jung Lee; Miye Kwon; Ilnam Kang; Eun-Gyoung Oh; Hong-Sik Yu; Soon-Bum Shin; Sang-Jong Kim

doi:10.1007/s12275-010-0047-4

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Research Support, Non-U.S. Gov't
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: Cheonghoon Lee ^1,2†, Sooryun Cheong ^1†, Hee-Jung Lee ³, Miye Kwon ¹, Ilnam Kang ^1,2, Eun-Gyoung Oh ³, Hong-Sik Yu ³, Soon-Bum Shin ³, Sang-Jong Kim ^1,2; Journal of Microbiology 2010;48(5):586-593.
DOI: https://doi.org/10.1007/s12275-010-0047-4
Published online: November 3, 2010

¹School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea, ²Institute of Microbiology, Seoul National University, Seoul 151-742, Republic of Korea, ³Food Safety Research Division, National Fisheries Research & Development Institute, Busan 619-705, Republic of Korea

Corresponding author: Sang-Jong Kim , Tel: +82-2-880-6704,

Received: 2 February 2010 • Accepted: 30 June 2010

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Abstract

Noroviruses (NoV) are the key cause of acute epidemic gastroenteritis, and oysters harvested from NoVpolluted 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 NoVseeded 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 QIAshredderTM 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.

Keywords: norovirus;oyster;nested RT-PCR;primer sensitivity and specificity;RNA extraction efficiency

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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

J. Microbiol. 2010;48(5):586-593. Published online November 3, 2010

DOI: https://doi.org/10.1007/s12275-010-0047-4

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