Background:  Rapid detection of HIV nucleic acids would be useful for detection of HIV-infected infants and for routine monitoring of therapeutic effectiveness. However, placement of this technology in low-resource settings is hampered by high cost per test, complexity of the testing protocols, and the technical expertise needed to perform the testing. Microfluidic technology permits the miniaturization of the basic functions of nucleic acids extraction, amplification, and detection in inexpensive, portable devices. In this study, DNA was extracted and specific HIV-1 sequences were amplified by polymerase chain reaction (PCR) using a credit-card–sized device and microfluidic principles for both nucleic acids extraction and amplification.

Methods:  HIV-1-uninfected whole blood was spiked with various concentrations of 8E5 cells, which contain a single, integrated copy of HIV-1 DNA. Using the microfluidic device and the QiAMP DNA blood mini-kit, we extracted 200 µL of the spiked specimens ranging from100,000 cells/mL to 100 cells/mL were extracted. Extracts were amplified using a double-stranded primer from the long terminal repeat region on the Stratagene MX 3000 for real-time detection of amplicons, or using equipment designed for the microfluidic cards. HIV-1 amplicons produced using the microfluidic protocols were detected by end-point fluorescence. HIV-1-infected (n=26) and HIV-uninfected (n=20) whole blood specimens were extracted and amplified by both methods.

Results:  The limit of detection for the 8E5 spiked extracts from the 2 devices was similar:  10 copy input for Micronics; 4 copy input for Qiagen. The threshold cycle for the initial detection of HIV-1 long terminal repeat amplicons was also similar between the 2 methods (10 copy input:  Micronics Ct = 32.35; Qiagen Ct = 31.87). All HIV-1-infected specimens extracted by both methods were detected with similar threshold cycles when amplified by the reference method. HIV-1-uninfected whole blood extracted by either method did not produce any amplified material. All of the HIV-1-infected whole blood specimens that were extracted and amplified by the microfluidic protocols were detected, while the HIV-1-uninfected specimens did not produce any amplified material.

Conclusions:  Microfluidic processing of whole blood specimens can effectively extract and amplify viral nucleic acids and could provide for point-of-care nucleic acids amplification testing in a variety of clinical venues. The level of sensitivity provides excellent qualitative detection of HIV-1 DNA in infected individuals.