5th International AIDS Society Conference on HIV Pathogenesis and Treatment Cape Town - July 19 - 22, 2009

ACCURATE QUANTIFICATION OF HIV-1 CDNA ON A CREDIT-CARD-SIZED MICROFLUIDIC PLATFORM

IAS Conf HIV Pathog Treat 2009 Jul 19-22;5th: Abstract No. MOPDB101

O.T. Ng¹, J. Pipper², S.Y. Wee ³, A. Chua⁴, Y.S. Leo³, J. Husak⁵, M. Martinkovic⁵, K.K. Chew³, M. Inoue^6
1Tan Tock Seng Hospital, Infectious Disease, Singapore, Singapore, ²Institute of Bioenginering and Nanotechnology, Singapore, Singapore, ³Tan Tock Seng Hospital, Singapore, Singapore, ⁴National University Hospital, Singapore, Singapore, ⁵Institute of Bioengineering and Nanotechnology, Singapore, Singapore, ⁶Institute of Cell and Molecular Biology, Singapore, Singapore

BACKGROUND: A point-of-care HIV-1 viral quantitation test for treatment monitoring is much needed, but unavailable (Calmy A, et al. Clin Infect Dis. 2007 Jan 1;44(1):128-34). We previously reported a disposable miniaturized lab-on-a-chip platform performing all conventional laboratory functions involving sample preparation and quantitative reverse-transcription real-time PCR assay for avian influenza detection (Pipper J, et al. Nat Med. 2007 Oct;13(10):1259-63). We have improved our current hand-held platform for HIV-1 quantitation to include multiplexing for internal controls (IC) to eliminate 'false negatives'. We compared our current platform for HIV-1 quantitation with a conventional real-time PCR system.

METHODS: We evaluated the performance of our current system using a 1-uL droplet containing 101 to 109 copies of HIV- 1 cDNA with 10 copies of IC per reaction (c/rxn). The assay was performed using in-house designed primers with fluorescent probe targeting the HIV-1 gag region. The reaction mix contained IC cDNA targets co-amplified by the primers for HIV-1 and detected by IC-specific fluorescent probes. Results were compared against parallel identical experiments using the Stratagene Mx 3000P real-time PCR system. Crossing-points were determined using Larionov's method (Larionov A, et al.. BMC Bioinformatics. 2005 Mar 21;6:62).

RESULTS: Target cycle-threshold results ranged on a log-linear scale from Ct 37.2 (10¹ c/rxn) to 8.9 (10⁹ c/rxn) for the hand-held device (R²=1.00) and Ct 36.9 (10¹ c/rxn) to 8.7 (10⁹ c/rxn) (R²=1.00) for the Stratagene. The IC was consistently detected excluding reaction inhibition. The correlation between the two devices was excellent (graph 1).

CONCLUSION: We demonstrate a hand-held device, which accurately quantifies HIV-1 cDNA using duplex real-time PCR. We are currently incorporating direct one-step blood sample preparation to actualize a field-ready device.

2009-07-22
MOPDB101
Poster Discussion MOPDB1 - Update on Diagnosing and Monitoring

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