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Development of Molecular Diagnostic System with High Sensitivity for the Detection of Human Sapovirus from Water Environments

Biomedical Science Letters 2021년 27권 1호 p.35 ~ 43
Lee Si-Won, 배경선, 이진영, 주윤리, 김지혜, 유경아,
소속 상세정보
 ( Lee Si-Won ) - Shinhan University Department of Biomedical Laboratory Science
배경선 ( Bae Kyung-Seon ) - National Institute of Environmental Research Water Supply and Sewerage Research Division
이진영 ( Lee Jin-Young ) - LSLK Co.
주윤리 ( Joo Youn-Lee ) - National Institute of Environmental Research Water Supply and Sewerage Research Division
김지혜 ( Kim Ji-Hae ) - National Institute of Environmental Research Water Supply and Sewerage Research Division
유경아 ( You Kyung-A ) - National Institute of Environmental Research Water Supply and Sewerage Research Division

Abstract


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.

키워드

Human Sapovirus; HuSaV; Positive control; SL® Non-specific reaction inhibitor; Water environments

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