대한의생명과학회지 (Biomedical Science Letters)

  • 제29권2호
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  • Pages.81-87
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  • 2023
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  • 1738-3226(pISSN)
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  • 2288-7415(eISSN)
대한의생명과학회 (The Korean Society for Biomedical Laboratory Sciences)
권호 표지
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Evaluation of Proposed Diagnostic System for Detection of Pan-enterovirus Using Reverse Transcription Nested PCR from Water Environment

  • Siwon Lee (R&D Team, LSLK Co. Ltd.) ;
  • Kyung Seon Bae (Water Supply and Sewerage Research Division, National Institute of Environmental Research) ;
  • Jin-Ho Kim (Institute of Tissue Regeneration Engineering (ITREN), Dankook University) ;
  • Ji-Hyun Park (Water Supply and Sewerage Research Division, National Institute of Environmental Research) ;
  • Ji Hye Kim (Water Supply and Sewerage Research Division, National Institute of Environmental Research) ;
  • Ji-Yeon Park (Department of Chemistry, College of Science and Engineering, Dankook University) ;
  • Kyung-Jin Lee (Department of Chemistry, College of Science and Engineering, Dankook University) ;
  • Chae-Rin Jeon (R&D Team, LSLK Co. Ltd.) ;
  • Jeong-Ki Yoon (Water Supply and Sewerage Research Division, National Institute of Environmental Research) ;
  • Soo-Hyung Lee (Water Supply and Sewerage Research Division, National Institute of Environmental Research) ;
  • Eung-Roh Park (Water Supply and Sewerage Research Division, National Institute of Environmental Research)
  • 투고 : 2023.03.29
  • 심사 : 2023.04.21
  • 발행 : 2023.06.30
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초록

Pan-Enterovirus (Pan-EV) infects millions of children and infants worldwide every year. As severe infections have recently been reported, the need for monitoring has consequently intensified. Pan-EV is a categorical name for waterborne enteroviruses belonging to the Picornaviridae family, and includes a wide range of pathogens including Coxsackievirus (CoxV), Echovirus (EcoV) and Enterovirus (EV). In this study, we proposed an optimal RT-nested PCR method for diagnosis of various types of Pan-EV in an aquatic environment and developed a positive control. Considering detection sensitivity, specific reaction, and final identification, one condition capable of amplifying 478 bp among the four candidates in the 1st round PCR (RT-PCR) and one condition in the 2nd round PCR (nested PCR) were selected. Through the detection of nucleic acids extracted from 123 groundwater samples and the detection sensitivity test based on artificial spiking in the sample, the methods are optimal for non-disinfected water samples such as groundwater. We developed a positive control for Pan-EV detection that can be amplified to different sizes under the two conditions. Accuracy could be further improved by testing for contamination from the control group. The method proposed in this study and the positive control developed are expected to be used in monitoring Pan-EV in aquatic environments including groundwater through future research using more samples.

키워드

과제정보

This work was supported by a grant from the National Institute of Environmental Research (NIER), funded by the Ministry of Environment (ME) of the Republic of Korea (NIER-2023-01-01-155). In addition, we would like to thank the three institutes (K-water, DK-EcoV and Shinhan University) for assisting with the validation of the methods.

참고문헌

  1. Bosch A. Human enteric viruses in the water environment: a minireview. Int Microbiol. 1998. 1: 191-196.
  2. Chiang PS, Huang ML, Luo ST, Lin TY, Tsao KC, Lee MS. Comparing molecular methods for early detection and serotyping of enteroviruses in throat swabs of pediatric patients. PLoS One. 2012. 7: e48269.
  3. International committee on taxonomy of viruses (ICTV). 2017. Master Species List 2018 (10th Report). Retrieved 2018-07-29.
  4. International committee on taxonomy of viruses (ICTV). 2019. Master Species List 2018 (10th Report). Retrieved 2019-02-19.
  5. Leclerc H, Edberg S, Pierzo V, Delattre JM. Bacteriophages as indicators of enteric viruses and public health risk in ground-waters. J Appl Microbiol. 2000. 88: 5-21. https://doi.org/10.1046/j.1365-2672.2000.00949.x
  6. Lee HK, Jeong YS. Comparison of total culturable virus assay and multiplex integrated cell culture-PCR for reliability of waterborne virus detection. Appl Environ Microbiol. 2004. 70: 3632-3636. https://doi.org/10.1128/AEM.70.6.3632-3636.2004
  7. Lee S, Bae KS, Lee JY, et al. Development of molecular diagnostic system with high sensitivity for the detection of human sapovirus from water environments. Biomed Sci Letters. 2021a. 27: 35-43. https://doi.org/10.15616/BSL.2021.27.1.35
  8. Lee S, Bae KS, Park J, et al. Mornitoring and identification of human astrovirus from groundwater in Korea based on highly sensitive RT-nested PCR primer sets. Biomed Sci Letters. 2021b. 27: 255-263. https://doi.org/10.15616/BSL.2021.27.4.255
  9. Moore BE. Survival of human immunodeficiency virus (HIV), HIV-infected lymphocytes, and poliovirus in water. Appl Environ Microbiol. 1993. 59: 1437-1443. https://doi.org/10.1128/aem.59.5.1437-1443.1993
  10. National Institute of Environmental Research (NIER). Development and Verification of Genetically Diagnostic Method for the Detection of Non-regulated Viruses from Water Environment (I). 2016. NIER.
  11. NIER. Development and Verification of Genetically Diagnostic Method for the Detection of Non-regulated Viruses from Water Environment. 2017. NIER
  12. US EPA, 2014. Method 1693: Cryptosporidium and Giardia in disnfected wastewater by concentration/IMS/IFA, EPA 821-R-14-013, Washington, D.C.
  13. Zoll GJ, Melchers WJ, H, et al. General primer-mediated polymerase chain reaction for detection of enteroviruses: application for diagnostic routine and persistent infections. J Clin Microbiol. 1992. 30: 160-165. https://doi.org/10.1128/jcm.30.1.160-165.1992