QUANTIFICATION OF HIV AND HCV VIRAL LOAD WITH LARGE DYNAMIC RANGE USING MULTIVOLUME DIGITAL REVERSE TRANSCRIPTION PCR ON A ROTATIONAL SLIPCHIP Feng Shen † , Bing Sun, Jason E. Kreutz, Elena K. Davydov, Wenbin Du ‡ , Rustem F. Ismagilov * Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East. 57th St., Chicago, Illinois 60637 † Current Address: SlipChip, LLC, 2201 Campbell Park Drive, Chicago, IL, 60612 ‡ Current Address: Department of Chemistry, Renmin University of China, Beijing, China, 100872 *Current Address: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125 ABSTRACT This presentation describes a SlipChip-based microfluidic platform capable of performing multiplex digital RT-PCR with large and tunable dynamic range, using a mathematical approach based on Most Probable Number theory. By using compartments of different volumes, we achieved a dynamic range of 520 – 4,000,000 molecules/mL at 3-fold resolution with 95% confidence interval. This SlipChip platform enables fast, accurate, low-cost, “instrument-free” quantification of RNA, crucial for resource-limited settings. We validated the device using deidentified clinical patient HIV viral RNA and results from the SlipChip were in good agreement with Roche COBAS® AmpliPrep / COBAS® TaqMan® HIV-1 Test, v2.0 system. We also expanded the device to allow for multiplexing and validated the multiplexed device using HIV and HCV viral RNA. KEY WORDS Digital RT-PCR, High dynamic range, multivolume, HIV viral load INTRODUCTION This presentation will describe a recently developed method[1] to quantify RNA using digital RT-PCR on a multivolume (MV) SlipChip platform. The HIV viral load test is a quantitative measurement of HIV RNA which provides important information in monitoring disease status and in guiding therapy. As antiretroviral treatments become more widely available, there is an ever-increasing demand to evaluate viral loads at regular intervals to prevent the spread of drug resistance. However, it remains difficult to do, particularly in resource-limited settings, because it requires the measurement of RNA over a large dynamic range (from 50 to 10 6 molecules/mL for HIV and up to 10 8 molecules/mL for HCV). The most commonly used method to quantify RNA is real-time quantitative Reverse-Transcription Polymerase Chain Reaction (RT-PCR), but it is usually only provided in centralized facilities with multiple instruments and highly skilled technicians, thus its application is not accessible in resource-limited settings. Compared with real-time RT-PCR, digital RT- PCR developed on microfluidic platforms has certain advantages such as low cost and small sample volume. However most of these microfluidic platforms still have two drawbacks: the need for relatively complex control systems to handle samples and the need for large numbers of experiments/compartments to obtain wide dynamic range and high resolution. To solve this problem, a rotational SlipChip with sets of compartments of different volumes was developed (Figure 1). The SlipChip is a microfluidic platform consisting of two plates that can manipulate liquid samples from pL-to µL-scales by relative movement of the plates without the need for complex control systems[2]. The SlipChip has been previously used for multiplex PCR[3], digital PCR[4], and digital isothermal amplification (RPA)[5]. Instead of the uniform well volumes of previous designs, this MV design used four sets of wells with volumes of 1 nL, 5 nL, 25 nL, and 125 nL respectively. The volume of the well determines the probability that molecules will be trapped in the well. When there is at least one molecule present, amplification generates a “yes” signal, otherwise a “no” signal will be observed. We demonstrated the quantification of HIV in archived, deidentified clinical patient samples with digital RT-PCR on this design. We then modified the design to incorporate multiplexing capability while maintaining the large dynamic range by adding additional well volumes. The multiplexed design could analyze five separate samples simultaneously; when this second design is used to analyze a single sample, it has the potential to reach an even higher dynamic range (40-20,000,000 molecules/mL)[1]. EXPERIMENTAL The procedure for fabricating the SlipChip was described in previous work[2]. The SlipChip was made from soda−lime glass plates coated with chromium and photoresist. By using a two-step exposure and etching procedure, patterns of two different depths were generated on the chip. After etching, the glass plate was silanized with dichlorodimethylsilane and assembled under 4:1v/v tetradecane: mineral oil under the stereomicroscope for precise alignment. A control RNA molecule (906 nucleotide) was synthesized from the LITMUS 28iMal Control Plasmid using a HiScribe™ T7 In Vitro Transcription Kit and purified using MinElute PCR purification. Plasma samples containing the HIV virus were obtained from archived, deidentified patient samples at the University of Chicago Hospital. Plasma containing a control HCV virus (25 million IU/mL) was purchased from AcroMetrix (Benicia, CA). All plasma samples were purified using iPrepTM purification. Primers for the control RNA (906 nt) were: GAA GAG TTG GCG AAA GAT CCA CG and CGA GCT CGA ATT AGT CTG CGC. 978-0-9798064-4-5/μTAS 2011/$20©11CBMS-0001 290 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 2-6, 2011, Seattle, Washington, USA