대한의생명과학회지 (Biomedical Science Letters)

  • 제27권1호
  • /
  • Pages.35-43
  • /
  • 2021
  • /
  • 1738-3226(pISSN)
  • /
  • 2288-7415(eISSN)
대한의생명과학회 (The Korean Society for Biomedical Laboratory Sciences)
권호 표지
DOI QR코드

DOI QR Code

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

  • Lee, Siwon (Department of Biomedical Laboratory Science, Shinhan University) ;
  • Bae, Kyung Seon (Water Supply and Sewerage Research Division, National Institute of Environmental Research) ;
  • Lee, Jin-Young (R&D Team, LSLK Co.) ;
  • Joo, Youn-Lee (Water Supply and Sewerage Research Division, National Institute of Environmental Research) ;
  • Kim, Ji-Hae (Water Supply and Sewerage Research Division, National Institute of Environmental Research) ;
  • You, Kyung-A (Water Supply and Sewerage Research Division, National Institute of Environmental Research)
  • 투고 : 2021.01.15
  • 심사 : 2021.03.15
  • 발행 : 2021.03.31
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

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

참고문헌

  1. Botes M, de Kwaadsteniet M, Cloete TE. Application of quantitative PCR for the detection of microorganisms in water. Anal Bioanal Chem. 2013. 405: 91-108. https://doi.org/10.1007/s00216-012-6399-3
  2. Bhullar SS, Chandak NH, Purohit H, et al. Determination of viral load by quantitative real-time PCR in herpes simplex encephalitis patients. Intervirology. 2014. 57: 1-7. https://doi.org/10.1159/000351521
  3. Cho KB. Development of nested PCR primer set for the specific and highly sensitive detection of human parvovirus B19. Biomed Sci Lett. 2018a. 24: 390-397. https://doi.org/10.15616/BSL.2018.24.4.390
  4. Cho KB. Construction of Improved PCR primer set for the detection of human enteric adenovirus 41. Biomed Sci Lett. 2018b. 24: 230-238. https://doi.org/10.15616/BSL.2018.24.3.230
  5. Cho SR, Lee DY, Jung S, et al. Pathogen surveillance of acute viral gastroenteritis in Korea in 2017. Public Health Weekly Report. 2018. 11: 1374-1380.
  6. Cho SR, Yun SJ, Chae SJ, et al. An outbreak associated with sapovirus GI.3 in an elementary school in Gyeonggi-do, Korea. J Korean Med Sci. 2020. 35: e281. https://doi.org/10.3346/jkms.2020.35.e281
  7. Dalecka B, Mezule L. Study of potential PCR inhibitors in drinking water for Escherichia coli identification. Agron Res. 2018. 16: 1351-1359.
  8. Fukuda S, Takao S, Kuwayama M, Shimazu Y, Miyazaki K. Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay. J Clin Microbiol. 2006. 44: 1376-1381. https://doi.org/10.1128/JCM.44.4.1376-1381.2006
  9. Hwnag BM, Lee DY, Chung GT, Yoo CK. Laboratory surveillance of viral acute gastroenteritis in Korea, 2014. Public Health Weekly Report. 2015. 8: 1172-1177.
  10. Khamrin P, Okame M, Thongprachum A, et al. A single-tube multiplex PCR for rapid detection in feces of 10 viruses causing diarrhea. J Virol Methods. 2011. 173: 390-393. https://doi.org/10.1016/j.jviromet.2011.02.012
  11. Kitajima M, Oka T, Haramoto E, et al. Detection and genetic analysis of human sapoviruses in river water in Japan. Appl Environ Microbiol. 2010. 76: 2461-2467. https://doi.org/10.1128/AEM.02739-09
  12. Korea Centers for Disease Control and Prevention (KCDC). Practical guidelines for laboratory diagnosis of waterborne foodborne diseases. 2015. pp. 88-89. KCDC, Chungcheongbuk-do, Korea.
  13. Korea Centers for Disease Control and Prevention (KCDC). Weekly sentinel surveillance report, PHWR. 2019. pp. 2283. KCDC, Chungcheongbuk-do, Korea.
  14. Korea Ministry of Food and Drug Safety (KMFDS). Test method of food poisoning cause investigation. 2015. pp. 223-227. KMFDS, Chungcheongbuk-do, Korea.
  15. Kumthip K, Khamrin P, Ushijima H, et al. Genetic recombination and diversity of sapovirus in pediatric patients with acute gastroenteritis in Thailand, 2010-2018. PeerJ. 2020. 8: e8520. https://doi.org/10.7717/peerj.8520
  16. Lee S. A study of molecular biological detection methods for seed-transmitted viruses in quarantine. Ph. D. thesis. 2013. Dankook University, Cheonan, Chungcheongnam-do, Korea.
  17. Lee SG, Lee SH, Park SW, et al. Standardized positive controls for detection of norovirus by reverse transcription PCR. Virol J. 2011. 260: 1-8.
  18. Liu X, Yamamoto D, Saito M, et al. Molecular detection and characterization of sapovirus in hospitalized children with acute gastroenteritis in the Philippines. J Clin Virol. 2015. 68: 83-88. https://doi.org/10.1016/j.jcv.2015.05.001
  19. Mano J, Hatano S, Futo S, et al. Development of direct real-time PCR system applicable to a wide range of foods and agricultural products. Shokuhin Eiseigaku Zasshi. 2014. 55: 25-33. https://doi.org/10.3358/shokueishi.55.25
  20. NIER. Development and verification of genetically diagnostic method for the detection of non-regulated viruses from water environment (I). 2016. pp. 1-21. NIER, Incheon, Korea.
  21. Oka T, Katayama K, Hansman GS, et al. Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction. J Med Virol. 2006. 78: 1347-1353. https://doi.org/10.1002/jmv.20699
  22. Oka T, Wang Q, Katayama K, Saif LJ. Comprehensive review of human sapoviruses. Clin Microbiol Rev. 2015. 28: 32-53. https://doi.org/10.1128/CMR.00011-14
  23. Ponchel F, Toomes C, Bransfield K, et al. Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnol. 2003. 3: 18. https://doi.org/10.1186/1472-6750-3-18
  24. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol. 2012. 113: 1014-1026. https://doi.org/10.1111/j.1365-2672.2012.05384.x
  25. Shigemoto N, Fukuda S, Tanizawa Y, et al. Detection of norovirus, sapovirus, and human astrovirus in fecal specimens using a multiplex reverse transcription-PCR with fluorescent dye-labeled primers. Microbiol Immunol. 2011. 55: 369-372. https://doi.org/10.1111/j.1348-0421.2011.00325.x
  26. Smith CJ, Osborn AM. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol. 2009. 67: 6-20. https://doi.org/10.1111/j.1574-6941.2008.00629.x
  27. Thwiny H, Hasony H. Molecular detection of human sapovirus from healthy and hospitalized children with acute gastroenteritis in Basrah. JIARM. 2015. 3: 393-403.