Amplification of Viral RNA from Drinking Water Using TransPlex™ Whole-transcriptome Amplification

Overview

Journal J Appl Microbiol

Publisher Oxford University Press

Date 2011 Apr 12

PMID 21477067

Citations 1

Authors

J K Parker

T-Y Chang

J S Meschke

Affiliations

Soon will be listed here.

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.

Citing Articles

Fidelity and representativeness of two isothermal multiple displacement amplification systems to preamplify limiting amounts of total RNA.

Gadkar V, Arseneault T, Filion M Mol Biotechnol. 2013; 56(4):377-85.

PMID: 24198216 DOI: 10.1007/s12033-013-9718-9.

References

Bergen A, Haque K, Qi Y, Beerman M, Garcia-Closas M, Rothman N . Comparison of yield and genotyping performance of multiple displacement amplification and OmniPlex whole genome amplified DNA generated from multiple DNA sources. Hum Mutat. 2005; 26(3):262-70. DOI: 10.1002/humu.20213. View

Sorensen K, Jespersgaard C, Vuust J, Hougaard D, Norgaard-Pedersen B, Andersen P . Whole genome amplification on DNA from filter paper blood spot samples: an evaluation of selected systems. Genet Test. 2007; 11(1):65-71. DOI: 10.1089/gte.2006.0503. View

Lauss M, Vierlinger K, Weinhaeusel A, Szameit S, Kaserer K, Noehammer C . Comparison of RNA amplification techniques meeting the demands for the expression profiling of clinical cancer samples. Virchows Arch. 2007; 451(6):1019-29. DOI: 10.1007/s00428-007-0522-4. View

Breitbart M, Rohwer F . Method for discovering novel DNA viruses in blood using viral particle selection and shotgun sequencing. Biotechniques. 2005; 39(5):729-36. DOI: 10.2144/000112019. View

Tomlins S, Mehra R, Rhodes D, Shah R, Rubin M, Bruening E . Whole transcriptome amplification for gene expression profiling and development of molecular archives. Neoplasia. 2006; 8(2):153-62. PMC: 1578511. DOI: 10.1593/neo.05754. View

Chen G, Kobayashi L, Nazarenko I . Effect of sample aliquot size on the limit of detection and reproducibility of clinical assays. Clin Chem. 2007; 53(11):1962-5. DOI: 10.1373/clinchem.2007.089854. View

Vora G, Meador C, Stenger D, Andreadis J . Nucleic acid amplification strategies for DNA microarray-based pathogen detection. Appl Environ Microbiol. 2004; 70(5):3047-54. PMC: 404398. DOI: 10.1128/AEM.70.5.3047-3054.2004. View

Gonzalez J, Portillo M, Saiz-Jimenez C . Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments. Environ Microbiol. 2005; 7(7):1024-8. DOI: 10.1111/j.1462-2920.2005.00779.x. View

Uda A, Tanabayashi K, Fujita O, Hotta A, Yamamoto Y, Yamada A . Comparison of whole genome amplification methods for detecting pathogenic bacterial genomic DNA using microarray. Jpn J Infect Dis. 2007; 60(6):355-61. View

10.

Berthet N, Reinhardt A, Leclercq I, van Ooyen S, Batejat C, Dickinson P . Phi29 polymerase based random amplification of viral RNA as an alternative to random RT-PCR. BMC Mol Biol. 2008; 9:77. PMC: 2535778. DOI: 10.1186/1471-2199-9-77. View

11.

Leclerc H, Schwartzbrod L, Dei-Cas E . Microbial agents associated with waterborne diseases. Crit Rev Microbiol. 2003; 28(4):371-409. DOI: 10.1080/1040-840291046768. View

12.

Panelli S, Damiani G, Espen L, Sgaramella V . Ligation overcomes terminal underrepresentation in multiple displacement amplification of linear DNA. Biotechniques. 2005; 39(2):174, 176, 178 passim. DOI: 10.2144/05392BM03. View

13.

Schwab K, de Leon R, Sobsey M . Concentration and purification of beef extract mock eluates from water samples for the detection of enteroviruses, hepatitis A virus, and Norwalk virus by reverse transcription-PCR. Appl Environ Microbiol. 1995; 61(2):531-7. PMC: 167314. DOI: 10.1128/aem.61.2.531-537.1995. View

14.

Van Gelder R, von Zastrow M, Yool A, Dement W, Barchas J, Eberwine J . Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci U S A. 1990; 87(5):1663-7. PMC: 53542. DOI: 10.1073/pnas.87.5.1663. View

15.

Wang E . RNA amplification for successful gene profiling analysis. J Transl Med. 2005; 3:28. PMC: 1201175. DOI: 10.1186/1479-5876-3-28. View

16.

Banach B, Orenstein J, Fox L, Randell S, Rowley A, Baker S . Human airway epithelial cell culture to identify new respiratory viruses: coronavirus NL63 as a model. J Virol Methods. 2008; 156(1-2):19-26. PMC: 2671689. DOI: 10.1016/j.jviromet.2008.10.022. View

17.

Gao H, Yang Z, Gentry T, Wu L, Schadt C, Zhou J . Microarray-based analysis of microbial community RNAs by whole-community RNA amplification. Appl Environ Microbiol. 2006; 73(2):563-71. PMC: 1796964. DOI: 10.1128/AEM.01771-06. View