해수 및 저질 시료에서의 흰반점바이러스(white spot syndrome virus) 검출을 위한 다양한 전처리 방법 평가
Evaluation of various extraction methods for white spot syndrome virus (WSSV) from seawater and sediment
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White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD), is the most significant viral pathogen infecting cultured shrimp. Because viruses are generally less abundant in environmental water and sediments contain PCR inhibitors, adequate extraction methods should be evaluated. This study compared various extraction methodologies to investigate WSSV in seawater and sediment to identify the optimal approach for transmission studies. To extract WSSV from sea- water, we concentrated WSSV using an iron flocculation assay and evaluated two methods: (i) iron flocculation with oxalate buffer suspension (I∙O method) and (ii) iron flocculation with direct elution (I∙D method). For sediment, we used sodium pyrophosphate and ethylenediaminetetraacetic acid buffer (S∙E method) and compared it with a commercial nucleic acid extraction kit. Furthermore, to enhance detection sensitivity, we evaluated a combination of the S∙E method with iron flocculation, referred to as the S∙E–I∙O method and the S∙E–I∙D method. Our findings suggest that the I∙D method for seawater samples provided a recovery rate of 91.22 ± 2.99% and a 1000-fold increase in detection sensitivity compared to direct nucleic acid extraction from seawater, which may aid in tracking WSSV dynamics in shrimp farm water. For sediment, the S∙E–I∙D method detected WSSV genome without the need for ultrasonication or ultracentrifugation, showing comparable efficacy to commercial kits. Notably, using the I∙O and S∙E–I∙O methods, further studies applying a viability qPCR technique to selectively amplify infectious pathogens could allow an accurate assessment of the WSSV decay rate within transmission reservoirs, which is an important epidemiological indicator of WSD.
Abstract
White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD), is the most significant viral pathogen infecting cultured shrimp. Because viruses are generally less abundant in environmental water and sediments contain PCR inhibitors, adequate extraction methods should be evaluated. This study compared various extraction methodologies to investigate WSSV in seawater and sediment to identify the optimal approach for transmission studies. To extract WSSV from sea- water, we concentrated WSSV using an iron flocculation assay and evaluated two methods: (i) iron flocculation with oxalate buffer suspension (I∙O method) and (ii) iron flocculation with direct elution (I∙D method). For sediment, we used sodium pyrophosphate and ethylenediaminetetraacetic acid buffer (S∙E method) and compared it with a commercial nucleic acid extraction kit. Furthermore, to enhance detection sensitivity, we evaluated a combination of the S∙E method with iron flocculation, referred to as the S∙E–I∙O method and the S∙E–I∙D method. Our findings suggest that the I∙D method for seawater samples provided a recovery rate of 91.22 ± 2.99% and a 1000-fold increase in detection sensitivity compared to direct nucleic acid extraction from seawater, which may aid in tracking WSSV dynamics in shrimp farm water. For sediment, the S∙E–I∙D method detected WSSV genome without the need for ultrasonication or ultracentrifugation, showing comparable efficacy to commercial kits. Notably, using the I∙O and S∙E–I∙O methods, further studies applying a viability qPCR technique to selectively amplify infectious pathogens could allow an accurate assessment of the WSSV decay rate within transmission reservoirs, which is an important epidemiological indicator of WSD.
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