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Amplification of viral RNA from drinking water using TransPlex™ whole‐transcriptome amplification
Author(s) -
Parker J.K.,
Chang T.Y.,
Meschke J.S.
Publication year - 2011
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2011.05029.x
Subject(s) - rna , biology , nucleic acid , transcriptome , reverse transcriptase , microbiology and biotechnology , serial dilution , dna , polymerase chain reaction , gene , genetics , gene expression , medicine , alternative medicine , pathology
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.