A Portable, Pressure Driven, Room Temperature Nucleic Acid Extraction and Storage System for Point of Care Molecular Diagnostics.

Many new and exciting portable HIV viral load testing technologies are emerging for use in global medicine. While the potential to provide fast, isothermal, and quantitative molecular diagnostic information to clinicians in the field will soon be a reality, many of these technologies lack a robust front end for sample clean up and nucleic acid preparation. Such a technology would enable many different downstream molecular assays. Here, we present a portable system for centrifuge-free room temperature nucleic acid extraction from small volumes of whole blood (70 µL), using only thermally stable reagents compatible with storage and transport in low resource settings. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis of simulated samples demonstrate a lower limit of detection of 1000 copies/ml, with the ability to detect differences in viral load across four orders of magnitude. The system can also be used to store extracted RNA on detachable cartridges for up to one week at ambient temperature, and can be operated using only hand generated air pressure.

[1]  K. A. Wolfe,et al.  Toward a microchip‐based solid‐phase extraction method for isolation of nucleic acids , 2002, Electrophoresis.

[2]  V. Soriano,et al.  Correlation between Human Immunodeficiency Virus Type 1 (HIV-1) RNA Measurements Obtained with Dried Blood Spots and Those Obtained with Plasma by Use of Nuclisens EasyQ HIV-1 and Abbott RealTime HIV Load Tests , 2009, Journal of Clinical Microbiology.

[3]  K. A. Wolfe,et al.  Microchip-based purification of DNA from biological samples. , 2003, Analytical chemistry.

[4]  Jerome P Ferrance,et al.  Chitosan-coated silica as a solid phase for RNA purification in a microfluidic device. , 2009, Analytical chemistry.

[5]  G. Aldrovandi,et al.  Multicenter Evaluation of Use of Dried Blood and Plasma Spot Specimens in Quantitative Assays for Human Immunodeficiency Virus RNA: Measurement, Precision, and RNA Stability , 2003, Journal of Clinical Microbiology.

[6]  Angelika Niemz,et al.  Point-of-care nucleic acid testing for infectious diseases. , 2011, Trends in biotechnology.

[7]  Feng Xu,et al.  Advances in developing HIV-1 viral load assays for resource-limited settings. , 2010, Biotechnology advances.

[8]  Andre Sharon,et al.  RNA isolation from mammalian cells using porous polymer monoliths: an approach for high-throughput automation. , 2010, Analytical chemistry.

[9]  Cheryl Jennings,et al.  HIV-1 Viral Load Assays for Resource-Limited Settings , 2006, PLoS medicine.

[10]  J. Landers,et al.  Evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format. , 2000, Analytical biochemistry.

[11]  Jerome P Ferrance,et al.  Dual-domain microchip-based process for volume reduction solid phase extraction of nucleic acids from dilute, large volume biological samples. , 2010, Analytical chemistry.

[12]  Lesley E Scott,et al.  Quantifying HIV for monitoring antiretroviral therapy in resource-poor settings. , 2010, The Journal of infectious diseases.

[13]  Jay L Gerlach,et al.  Cost analysis of centralized viral load testing for antiretroviral therapy monitoring in Nicaragua, a low-HIV prevalence, low-resource setting , 2010, Journal of the International AIDS Society.

[14]  L. Lynen,et al.  Monitoring for treatment failure in patients on first-line antiretroviral treatment in resource-constrained settings , 2010, Current opinion in HIV and AIDS.

[15]  Catherine M. Klapperich,et al.  Microfluidics-based extraction of viral RNA from infected mammalian cells for disposable molecular diagnostics , 2008 .

[16]  S. Lawn,et al.  Utility of CD4 cell counts for early prediction of virological failure during antiretroviral therapy in a resource-limited setting , 2008, BMC infectious diseases.

[17]  P. Mee,et al.  Evaluation of the WHO criteria for antiretroviral treatment failure among adults in South Africa , 2008, AIDS.

[18]  P. Yager,et al.  Point-of-care diagnostics for global health. , 2008, Annual review of biomedical engineering.

[19]  Jerome P Ferrance,et al.  DNA extraction using a tetramethyl orthosilicate-grafted photopolymerized monolithic solid phase. , 2006, Analytical chemistry.

[20]  Steven A Soper,et al.  96-well polycarbonate-based microfluidic titer plate for high-throughput purification of DNA and RNA. , 2008, Analytical chemistry.

[21]  N. Ford,et al.  HIV viral load monitoring in resource-limited regions: optional or necessary? , 2007, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[22]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[23]  J. Crump,et al.  Evaluation of a dried blood spot HIV-1 RNA program for early infant diagnosis and viral load monitoring at rural and remote healthcare facilities , 2009, AIDS.

[24]  Helen H. Lee,et al.  Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings. , 2007, The Analyst.

[25]  C. Klapperich,et al.  Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics. , 2006, Analytical chemistry.

[26]  D. Brambilla,et al.  Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma by Using Blood Dried on Filter Paper , 1998, Journal of Clinical Microbiology.

[27]  M. Salimans,et al.  Rapid and simple method for purification of nucleic acids , 1990, Journal of clinical microbiology.

[28]  Jerome P Ferrance,et al.  Chitosan as a polymer for pH-induced DNA capture in a totally aqueous system. , 2006, Analytical chemistry.

[29]  Jerome P Ferrance,et al.  Microchip-based macroporous silica sol-gel monolith for efficient isolation of DNA from clinical samples. , 2006, Analytical chemistry.

[30]  Ji Yoon Kang,et al.  A polymer lab-on-a-chip for reverse transcription (RT)-PCR based point-of-care clinical diagnostics. , 2008, Lab on a chip.

[31]  Jerome P Ferrance,et al.  Microchip‐Based Cell Lysis and DNA Extraction from Sperm Cells for Application to Forensic Analysis , 2006, Journal of forensic sciences.

[32]  C. Klapperich,et al.  Cell lysis and DNA extraction of gram-positive and gram-negative bacteria from whole blood in a disposable microfluidic chip. , 2009, Lab on a chip.

[33]  Jerome P Ferrance,et al.  Microfluidic-based DNA purification in a two-stage, dual-phase microchip containing a reversed-phase and a photopolymerized monolith. , 2007, Analytical chemistry.

[34]  James P Landers,et al.  Microchip-based solid-phase purification of RNA from biological samples. , 2008, Analytical chemistry.

[35]  Bernhard H Weigl,et al.  Laboratory operations, specimen processing, and handling for viral load testing and surveillance. , 2010, The Journal of infectious diseases.

[36]  C. Batt,et al.  Nucleic acid purification using microfabricated silicon structures. , 2003, Biosensors & bioelectronics.

[37]  M. Komiyama,et al.  Hydrolysis of DNA and RNA by lanthanide ions: mechanistic studies leading to new applications , 1999 .