Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp

Susan D Wharam,William H Wilson,Matthew J Hall

doi:10.1186/1743-422x-4-52

Abstract

BackgroundSignal-Mediated Amplification of RNA Technology (SMART) is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression) or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus.ResultsHere we report that the SMART assay allowed us to differentiate between infected and non-infected host cultures. Expression of the cyanophage strain S-PM2 portal vertex gene (g20) was detected from infected host Synechococcus sp. WH7803 cells. Using the SMART assay, we demonstrated that g20 mRNA peaked 240 – 360 minutes post-infection, allowing us to characterise this as a mid to late transcript. g20 DNA was also detected, peaking 10 hours post-infection, coinciding with the onset of host lysis.ConclusionThe SMART assay is based on isothermal nucleic acid amplification, allowing the detection of specific sequences of DNA or RNA. It was shown to be suitable for differentiating between virus-infected and non-infected host cultures and for the detection of virus gene expression: the first reported use of this technology for such applications.

Highlights

Signal-Mediated Amplification of RNA Technology (SMART) is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA or DNA
We have previously reported that the assay discriminated between similar g20 target DNA sequences from two different marine cyanophage strains [4]
In order to detect S-PM2 g20 mRNA from infected host cells, RNA and DNA were extracted from infected Synechococcus sp

Summary

Introduction

Signal-Mediated Amplification of RNA Technology (SMART) is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression) or DNA. The assay uses two oligonucleotide probes which hybridise to the target, at adjacent sites, and to each other to (page number not for citation purposes). When specific target nucleic acid is present, a T7 RNA polymerase promoter sequence within the 3WJ structure becomes double stranded, and activated. T7 RNA polymerase produces large amounts of an RNA transcript. This RNA is a RNA signal the assay signal and it can be further amplified by the same process if required, and detected by an enzymelinked oligosorbant assay (ELOSA) (Fig. 1b). Amplification and signal detection processes have been fully described and explained previously [1,4]

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Results

Conclusion