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KMID : 1144820210270010035
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대한의생명과학회지
2021 Volume.27 No. 1 p.35 ~ p.43
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Development of Molecular Diagnostic System with High Sensitivity for the Detection of Human Sapovirus from Water Environments
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Lee Si-Won
Bae Kyung-Seon
Lee Jin-Young
Joo Youn-Lee
Kim Ji-Hae
You Kyung-A
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Abstract
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Human Sapovirus (HuSaV) is one of the major causes of acute gastroenteritis in humans, and it is used as a molecular diagnostic technique based on polymerase chain reaction (PCR) from humans, food, shellfish, and aquatic environments. In this study, the HuSaV diagnosis technique was used in an aquatic environment where a number of PCR inhibitors are included and pathogens, such as viruses, are estimated to exist at low concentration levels. HuSaV-specific primers are improved to detect 38 strains registered in the National Center for Biotechnology Information (NCBI). The established optimal condition and the composition, including the RT-nested PCR primers and SL® Non-specific reaction inhibitor, were found to have 100 times higher sensitivity based on HuSaV plasmid than the previously reported methods (100 ag based on HuSaV plasmid 1 ng/μL). Through an artificial infection test, the developed method was able to detect at least 1 fg/μL of HuSaV plasmid contaminated with total nucleic acid extracted from groundwater. In addition, RT-nested PCR primer sets for HuSaV detection can react, and a positive control is developed to verify false positives. This study is expected to be used as a HuSaV monitoring method in the future and applied to the safety response to HuSaV from water environments.
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KEYWORD
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Human Sapovirus, HuSaV, Positive control, SL® Non-specific reaction inhibitor, Water environments
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