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. 2011 Jul;111(1):216-23.
doi: 10.1111/j.1365-2672.2011.05029.x. Epub 2011 May 5.

Amplification of viral RNA from drinking water using TransPlex™ whole-transcriptome amplification

J K Parker  1 , T-Y Chang, J S Meschke
Affiliations

Amplification of viral RNA from drinking water using TransPlex™ whole-transcriptome amplification

J K Parker et al. J Appl Microbiol. 2011 Jul.

Abstract

Aims: Viral pathogens in environmental media are generally highly diffuse, yet small quantities of pathogens may pose a health risk. This study evaluates the ability of TransPlex™ whole transcriptome amplification (WTA) to amplify small quantities of RNA viruses from complex environmental matrices containing background nucleic acids.

Methods and results: DNA extracts from mock drinking water samples containing mixed microbial populations were spiked with small quantities of echovirus type 13 (EV) RNA. Samples were amplified using a Transplex™ WTA kit, and EV-specific quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify target pathogens before and after application of WTA. Samples amplified by WTA demonstrated a decreased limit of detection. The log-linear relationship between serial dilutions was maintained following amplification by WTA.

Conclusions: WTA is able to increase the quantity of target organism RNA in mixed populations, while maintaining log linearity of amplification across different target concentrations.

Significance and impact of the study: WTA may serve as an effective preamplification step to increase the levels of RNA prior to detection by other molecular methods such as PCR, microarrays and sequencing.

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Figures

Figure 1
Figure 1
Experimental design.
Figure 2
Figure 2
Linear regressions of average echovirus pre‐WTA and post‐WTA C t values for background experiments. A 1 : 1 C t ratio line is shown for reference. BG 104×; ⋄ BG 103×; ▮ Control (no competing nucleic acid).
Figure 3
Figure 3
Linear regressions of average echovirus pre‐WTA and post‐WTA C t values for surface water experiments. A 1 : 1 C t ratio line is shown for reference. △ SW 105×; ◆ SW 104×; □ Control (no competing nucleic acid).
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