Tan Le Van, Van Doorn H Rogier, Van der Hoek Lia, Minh Hien Vo, Jebbink Maarten F, Quang Ha Do, Farrar Jeremy, Van Vinh Chau Nguyen, de Jong Menno D
Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City,
J Infect Dev Ctries. 2011 Mar 2;5(2):142-8. doi: 10.3855/jidc.1087.
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.
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.
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.
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.
基于cDNA-AFLP的病毒发现技术（VIDISCA）是一种不依赖序列的病毒发现方法，该方法最近被开发出来，并成功用于鉴定细胞培养物中的未知病毒。然而，其适用性受到低灵敏度的限制。
我们评估了通过随机PCR（rPCR）对靶序列进行预扩增是否能提高该方法的灵敏度，从而改善其在临床标本中的应用。此外，在实验方案中增加了超速离心步骤，以便合并多个样本，从而提高VIDISCA的分析通量。
我们发现，对于不同临床样本中的四种病毒中的两种，rPCR将VIDISCA的灵敏度提高了100倍，并且超速离心步骤能够分析大量（4毫升）样本，并同时处理多个（约40个）临床标本。
我们得出结论，在没有超深度测序平台的情况下，这种改良的VIDISCA方案是一种相对简便的方法，可用于筛查大量疑似含有先前未识别病原体的临床样本。
