title: Development of a low-resource RNA extraction cassette based on surface tension valves.
abstract: Nucleic acid-based diagnostics are highly sensitive and specific, but are easily disrupted by the presence of interferents in biological samples. In a laboratory or hospital setting, the influence of these interferents can be minimized using an RNA or DNA extraction procedure prior to analysis. However, in low-resource settings, limited access to specialized instrumentation and trained personnel presents challenges that impede sample preparation. We have developed a self-contained nucleic acid extraction cassette suitable for operation in a low-resource setting. This simple design contains processing solutions preloaded within a continuous length of 1.6 mm inner diameter Tygon tubing. Processing solutions are separated by air gaps and held in place during processing by the surface tension forces at the liquid-air interface, viz. surface tension valves. Nucleic acids preferentially adsorbed to silica-coated magnetic particles are separated from sample interferents using an external magnet to transfer the nucleic acid biomarker through successive solutions to precipitate, wash and elute in the final cassette solution. The efficiency of the extraction cassette was evaluated using quantitative reverse transcriptase PCR (qRT-PCR) following extraction of respiratory syncytial virus (RSV) RNA. RNA was recovered from TE buffer or from lysates of RSV infected HEp-2 cells with 55 and 33% efficiency, respectively, of the Qiagen RNeasy kit. Recovery of RSV RNA from RSV infected HEp-2 cells was similar at 30% of the RNeasy kit. An overall limit of detection after extraction was determined to be nearly identical (97.5%) to a laboratory-based commercially available kit. These results indicate that this extraction cassette design has the potential to be an effective sample preparation device suitable for use in a low-resource setting.
authors: Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee 37235, USA.
authors: Hali
authors: H
authors: Bordelon
authors: Nicholas M
authors: NM
authors: Adams
authors: Amy S
authors: AS
authors: Klemm
authors: Patricia K
authors: PK
authors: Russ
authors: John V
authors: JV
authors: Williams
authors: H Keipp
authors: HK
authors: Talbot
authors: David W
authors: DW
authors: Wright
authors: Frederick R
authors: FR
authors: Haselton
references: Trends Biotechnol. 2011 May;29(5):240-50
references: 21377748
references: J Mol Diagn. 2010 Sep;12(5):620-8
references: 20581047
references: Lab Chip. 2011 Mar 7;11(5):957-61
references: 21152489
references: Biomed Microdevices. 2010 Aug;12(4):705-19
references: 20401537
references: Appl Environ Microbiol. 1997 Oct;63(10):3741-51
references: 9327537
references: J Virol Methods. 1990 Feb;27(2):203-9
references: 2318929
references: Mol Biotechnol. 2004 Feb;26(2):133-46
references: 14764939
references: J Clin Microbiol. 1997 Apr;35(4):995-8
references: 9157172
references: Biochemistry. 1979 Nov 27;18(24):5294-9
references: 518835
references: Lab Chip. 2009 Sep 7;9(17):2484-94
references: 19680574
references: Ann Biomed Eng. 2006 Nov;34(11):1778-85
references: 17031592
references: Methods Enzymol. 1987;152:219-27
references: 2443794
references: J Clin Microbiol. 2005 Nov;43(11):5541-6
references: 16272483
references: J Med Virol. 2003 Jan;69(1):132-44
references: 12436489
references: Lab Chip. 2011 May 21;11(10):1747-53
references: 21423999
references: J Clin Microbiol. 2003 Jan;41(1):149-54
references: 12517840
references: Anal Biochem. 1987 Apr;162(1):156-9
references: 2440339