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Comparison of Direct RT-PCR, Cell Culture RT-PCR and Cell IFA for Viability and Infectivity Assay of Cryptosporidium

크립토스포리디움 활성 및 감염성 판정을 위한 direct RT-PCR, cell culture RT-PCR 및 cell culture IFA의 비교

  • Published : 2006.08.30

Abstract

Cryptosporidium is a waterborne pathogenic parasite which causes diarrhea. Immunomagnetic separation-immunofluorescent assay (IMS-IFA) has been a widely adopted for Cryptosporidium detection as standard method. However, this method does not provide information about viability or infectivity of Cryptosporidium. Therefore, many researchers have studied viability or infectivity analyses of Cryptosporidium with various methods such as vital staining, in vitro excystation, RT-PCR, cell culture, and mouse infection assay. In this study, two direct RT-PCR methods, cell culture RT-PCR and cell culture IFA were compared for sensitivity and other characteristics. The results showed that direct RT-PCR method with HSP70 genes had the highest sensitivity with detection up to 1 viable cell of Cryptosporidium. The infectious Cryptosporidium were detected up to 10 to 25 cells by cell culture methods in combination with RT-PCR and IFA. The infectious Cryptosporidium were apt to be quantified by cell culture IFA.

크립토스포리디움의 활성 및 감염성 판정을 위해 Direct RT-PCR, 세포배양 후 RT-PCR 및 면역형광염색법을 비교한 결과는 다음과 같다. 1) 크립토스포리디움의 HSP70 gene에 대해 direct RT-PCR한 결과, 민감도가 매우 높아 저농도로 존재하는 환경시료에서의 크립토스포리디움 활성을 모니터링하는데 장점이 있을 것으로 보이나, 감염성의 판정은 알 수 없으며, 정량화가 안되는 단점이 있었다. 2) ${\beta}-tubulin$ gene에 대해 RT-nested PCR을 한 결과 크립 토스포리디움의 난포낭이 $1{\times}10^4$ 세포수정도 되어야 검출이 되는 것으로 나타나 HSP70 gene에 대한 RT-PCR결과와 비교할 때 10,000배 이상 민감도가 떨어지는 것으로 나타났다. 3) 세포배양 후 RT-PCR 또는 면역형광염색법을 이용할 경우에는 민감도가 direct RT-PCR보다 다소 떨어지는 단점이 있었으나 크립토스포리디움의 오염원이나 오염이 심한 지역의 감염성 조사에 적합할 것으로 나타났으나, 정량화가 필요한 경우에는 세포배양 후 면역형광염색법이 효과적일 것으로 나타났다.

Keywords

References

  1. Bukhari, Z., M. M. Marshall, D. G. Korich, C. R. Fricker, H. V. Smith, J. Rosen and J. L. Clancy. 2000. Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts. Appl. Env. Microbiol. 66, 2972-2980 https://doi.org/10.1128/AEM.66.7.2972-2980.2000
  2. Campbell, A. T., L. J. Robertson and H. V. Smith. 1992. Viability of Cryptosporidium parvum outcasts: Correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes. Appl. Env. Microbiol. 58, 3488-3493
  3. Carey, C. M., H. Lee, J. T. Trevors. 2004. Biology, persistence and detection of Cryptosporidium parvum and Cryptosporidium hominis oocyst. Water Research 38, 818-862 https://doi.org/10.1016/j.watres.2003.10.012
  4. EPA. 2001. EPA 1623 Method : Cryptosporidium and Giardia in water by filtration/IMS/FA
  5. Jenkins, M., J. M. Trout, J. Higgins, M. Dorsch, D. Veal, R. Fayer. 2003. Comparison of tests for viable and infectious Cryptosporidium parvum oocysts. Parasitol Res. 89, 1-5 https://doi.org/10.1645/0022-3395(2003)089[0001:ROESIN]2.0.CO;2
  6. Michael, B. J., J. A. Lynne, D. B. Dwight, J. W. Mark and C. G. William. 1997. Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum outcasts. Appl. Env. Microbiol. 63, 3844-3850
  7. Rochelle, P. A., D. M. Ferguson, T. J. Handojo, R. D. Leon, M. H. Stewart and R. L. Wolfe. 1997. An assay combining cell culture with reverse transcriptase PCR to detect and determine the infectivity of waterborne Cryptosporidium parvum. Appl. Env. Microbiol. 63, 2029-2037
  8. Rochelle, P. A., M. M. Marshall, J. R. Mead, A. M. Johnson, D. G. Korich, J. S. Rosen, R. D. Leon. 2002. Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum. Appl. Env. Microbiol. 68, 3809-3817 https://doi.org/10.1128/AEM.68.8.3809-3817.2002
  9. Slifko, T. R., D. Friedman, J. B. Rose. 1997. An in vitro method for detecting infectious Cryptosporidium oocysts with cell culture. Appl. Env. Microbiol. 63, 3669-3675
  10. Widmer, G., E. A. Orbacz and S. Tzipori. 1999. ${\beta}-Tubulin$ mRNA as a marker of Cryptosporidium parvum oocyst viability. Appl. Env. Microbiol. 65, 1584-1588