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Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine
Susan D Wharam1, Matthew J Hall, William H Wilson
1Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine 04575, USA. swharam@bigelow.org
Background:
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. 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.
Results:
Here 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.
Conclusion:
The 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.
