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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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Abstract

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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Authors and Affiliations

  1. School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, 151-742, Republic of Korea

    Cheonghoon Lee, Sooryun Cheong, Miye Kwon, Ilnam Kang & Sang-Jong Kim

  2. Institute of Microbiology, Seoul National University, Seoul, 151-742, Republic of Korea

    Cheonghoon Lee, Ilnam Kang & Sang-Jong Kim

  3. Food Safety Research Division, National Fisheries Research & Development Institute, Busan, 619-705, Republic of Korea

    Hee-Jung Lee, Eun-Gyoung Oh, Hong-Sik Yu & Soon-Bum Shin

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  1. Cheonghoon Lee
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  2. Sooryun Cheong
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  3. Hee-Jung Lee
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  4. Miye Kwon
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  5. Ilnam Kang
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  7. Hong-Sik Yu
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  8. Soon-Bum Shin
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  9. Sang-Jong Kim
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Correspondence to Sang-Jong Kim.

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These authors contributed equally to this work.

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Lee, C., Cheong, S., Lee, HJ. et al. 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. 48, 586–593 (2010). https://doi.org/10.1007/s12275-010-0047-4

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

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