Zika Virus Amplification Using Strand Displacement Isothermal Method and Sequencing Using Nanopore Technology

Abstract

Development of novel point of care diagnostic methods in order to help in implementing disease control program and identifying the causative agent of an outbreak is crucial. Classical diagnostic techniques, e.g., real-time polymerase chain reaction (PCR), rely on the presence of the nucleic acid sequence of the target in GenBank. In the case of an emerging new strain of a known or novel pathogen, false-negative results will be recorded by PCR. On the other hand, next-generation sequencing technologies allow rapid whole genome sequencing without previous knowledge of the target. One of these methods is the Oxford Nanopore sequencing technique, which utilizes a portable device named MinION and has a short run time. In this protocol, we describe the development of a novel nanopore sequencing protocol by combining random isothermal amplification technology and nanopore sequencing. The established protocol is rapid (<7 h) and sensitive as less than 4% of the sequenced RNA belonged to the target virus, Zika. Interestingly, we have established an offline BLAST search for the data analysis that facilitates the use of the whole protocol at remote settings without the need of an Internet connection.