Random PCR and ultracentrifugation increases sensitivity and throughput of VIDISCA for screening of pathogens in clinical specimens

  • Le Van Tan Oxford University Clinical Research Unit, 190 Ben Ham Tu, District 5, Ho Chi Minh City, Vietanm
  • H. Rogier van Doorn Oxford University Clinical Research Unit, 190 Ben Ham Tu, District 5, Ho Chi Minh City, Vietanm; Centre for Tropical Medicine, Oxford University, U.K
  • Lia van der Hoek Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
  • Vo Minh Hien Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam
  • Maarten F. Jebbink Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
  • Do Quang Ha Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam
  • Jeremy Farrar Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam; Centre for Tropical Medicine, Oxford University, U.K
  • Nguyen Van Vinh Chau Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam
  • Menno D. de Jong Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam; Centre for Tropical Medicine, Oxford University, U.K; Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam

Abstract

Introduction: Virus discovery based on cDNA-AFLP (VIDISCA) is a sequence-independent virus discovery method that was recently developed and successfully used to characterize unknown viruses in cell cultures. Its applicability, however, is limited by its low sensitivity.

Methodology: We evaluated whether the introduction of prior amplification of target sequences by random PCR (rPCR) increases the sensitivity of this method to improve its use on clinical specimens. In addition, ultracentrifugation was added to the protocol to allow for pooling of multiple samples, thereby increasing analytical throughput of the VIDSCA.

Results: We showed that rPCR enhanced the sensitivity of VIDISCA by 100-fold for two out of four viruses in different clinical samples, and that the ultracentrifugation step allowed for analyzing samples of large volumes (4 ml) and simultaneous processing of multiple (~40) clinical specimens.

Conclusions: We conclude that this modified VIDISCA protocol is a relatively easy method to use for screening of large numbers of clinical samples that are suspected to contain previously unrecognized pathogens, in settings where ultradeep sequencing platforms are not available.

Published
2010-07-19
How to Cite
Tan L, van Doorn H, van der Hoek L, Minh Hien V, F. Jebbink M, Quang Ha D, Farrar J, Van Vinh Chau N, D. de Jong M (2010) Random PCR and ultracentrifugation increases sensitivity and throughput of VIDISCA for screening of pathogens in clinical specimens. The Journal Of Infection In Developing Countries 5 (02): 142-148. https://doi.org/10.3855/jidc.1087
Section
Technical Notes

Keywords

random PCR; VIDISCA; ultracentrifugatiaon; virus discovery