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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.

Type

Journal

Journal of infection in developing countries

Publication Date

02/2011

Volume

5

Pages

142 - 148

Addresses

Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.tanlv@oucru.org

Keywords

Humans, Viruses, Virus Diseases, Deoxyribonucleases, Type II Site-Specific, DNA, Complementary, DNA, Viral, RNA, Viral, Mass Screening, Ultracentrifugation, Sensitivity and Specificity, Polymerase Chain Reaction, Virology, Base Sequence, Molecular Sequence Data, Amplified Fragment Length Polymorphism Analysis