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The sequence-independent, single-primer amplifi cation (SISPA) enables the random amplifi cation of nucleic acids, allowing the detection and genome sequencing of diff erent viral agents. This feature of SISPA method provides evidence for application of it in monitoring the presence of adventitious RNA viruses in cell cultures. We evaluated SISPA method for the detection of a challenge RNA virus representing adventitious agent in cell cultures. Besides, by optimizing the SISPA method in our laboratory, we found false-positive results on negative control lanes in electrophoresis gels. To investigate the sources of contamination, false-positive results of SISPA were cloned into Escherichia coli cells, sequenced, and phylogenetically analyzed. This data revealed that the SISPA method can be used as an adjunct method to confi rm the absence of unexpected adventitious RNA viruses in cell cultures. The phylogenetic analysis of SISPA contaminant sequences showed that the false-positive results were caused by nucleic acid amplifi cation of commercial cDNA synthesis kit reagents, probably tracing back to expression plasmids and host ribosomal sequences, used for the production of enzymes. Therefore, laboratories using random amplifi cation methods must be constantly aware of the potentials of such contaminations, yielding false-positive results and background noise in the fi nal NGS reads.

archives of biotechnology and biomedicine

Sequence-independent single-primer-amplification (SISPA) as a screening technique for detecting unexpected RNA viral adventitious agents in cell cultures