SD-chip enabled quantitative detection of HIV RNA using digital nucleic acid sequence-based amplification (dNASBA)

Abstract

Quantitative detection of RNA is important in molecular biology and clinical diagnostics. Nucleic acid sequence-based amplification (NASBA), a single-step method to amplify single-stranded RNA, is attractive for use in point-of-care (POC) diagnostics because it is an isothermal technique that is as sensitive as RT-PCR with a shorter reaction time. However, NASBA is limited in its ability to provide accurate quantitative information, such as viral load or RNA copy number. Here we test a digital format of NASBA (dNASBA) using a self-digitization (SD) chip platform, and apply it to quantifying HIV-1 RNA. We demonstrate that dNASBA is more sensitive and accurate than the real-time quantitative NASBA, and can be used to quantify HIV-1 RNA in plasma samples. Digital NASBA is thus a promising POC diagnostics tool for use in resource-limited settings.

Figure 1.. NASBA.

(A) Schematic of RNA amplification in NASBA and generation of fluorescence. (B) qNASBA curves for reactions at HIV-1 RNA concentrations of 0 (black) and 1000 (red) copies/μL. (C) Electrophoresis of samples described in (B). (D) Plot of the time to positivity (TTP, the time required for fluorescence intensity to pass a threshold) versus input HIV-1 RNA concentration. The black square represents the TTP in each qNASBA. The black circle represents the average TTP in indicated concentration of HIV-1 RNA. At low HIV-1 RNA concentrations (≤10 copies/μL), TTP values varied widely and qNASBA could not accurately quantify RNA concentration. At higher concentrations (≥20 copies/μL), TTP showed a strong correlation with input concentration (R = 0.92). Error bars indicate standard deviation (n=3).

Figure 2.. Self-digitization chip.

(A) Schematic of SD chip. A 16 × 64 array of 6.5 nL microwells connected by channels is embedded in PDMS and covered with a PDMS-coated glass slide. (B) Photograph of SD chip. Enlarged area shows a section of the microwell array.

Figure 3.. Sample Loading.

(A) Schematic of sample loading onto the SD chip. Aqueous sample was added to the inlet, and a vacuum pump was connected to a PDMS adapter. (B) Schematic of SD chip filling. i) Branched drainage channels connecting individual chambers and channels were introduced to improve oil drainage from chambers; ii) The array is primed with oil mixture to eliminate air; iii) Aqueous sample travels from the inlet through the channels and expands into the chambers to lower the surface energy. Drainage channels allow oil to drain from chambers but are too small to allow passage of aqueous sample; iv) Additional oil displaces aqueous solution from channels but not from chambers, digitizing the sample. (C) Photographs of SD chip (1) empty, (2) filled with oil mixture, and (3) after digitization.

Figure 4.. Digital NASBA.

Digital NASBA results for different concentrations of HIV-1 RNA. (A) SD chip images. Positive chambers appear dark. (B) Measured versus input HIV-1 RNA concentration. Error bars indicate standard deviation (n=3).

Figure 5.

Quantification of HIV-1 RNA in human plasma using dNASBA (y-axis) and Abbot RealTime PCR assay (x-axis). The HIV-1 with different dilution factor (black dot, diluted 1, 10 and 100 times) was tested and HIV-2 was used as a control. Green error bars indicate standard deviation by Abbott RealTime assay, and red error bars indicate standard deviation by dNASBA (n=3).