An improved nucleic acid sequence-based amplification method mediated by T4 gene 32 protein

Abstract

The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes. Despite reports of successful enhancement of other DNA and RNA amplification methods using DNA and RNA binding proteins, this has not been reported for NASBA. Here, three single-stranded binding proteins, RecA, Extreme Thermostable Single-stranded binding protein (ET SSB) and T4 gene gp32 protein (gp32), were incorporated in NASBA protocol and used for single pot, one-step NASBA at 41 °C. Indeed, all SSBs showed significantly improved amplifications compared with the 2-step process, but only gp32 showed no non-specific aberrant amplification, and slightly improved the time-to-positivity in comparison with the conventional NASBA. For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.

Fig 1. A schematic representation of conventional NASBA and SSB mediated NASBA.

The conventional NASBA protocol requires a hybridization step before the addition of thermolabile NASBA enzymes to initiate the amplification and signal acquisition measuring the fluorescence intensity of molecular beacon. In single stranded binding protein mediated NASBA, the thermal denaturation-hybrization step is circumvented, allowing truly isothermal, single pot NASBA. In the graphic representation of the amplification cycle, the solid lines represent DNA, the wavey lines RNA.

Fig 2. The effect of SSBs on NASBA of the synthetic HIV-1 RNA gene using (a) 80 μg ET SSB, (b) 2 μg RecA and (c) 1 μg gp32 of a sample containing 2 x 10⁵ copies of synthetic HIV-1 RNA (~C_t 23) using the HIV-1 NASBA primer-probe set (S1 File).

All graphs show conventional 2-step NASBA (red), 1-step NASBA (black dash/dot), 1-step NASBA with SSB (blue dashed), and NTC in absence (solid black) and presence (solid blue) of SSB (n = 3).

Fig 3

(a) Effect of gp32 protein on the ttp for HIV-1 using NASBA and HIV-1 primer set (S1 File) (n = 3). (a) Optimization of gp32 using 0, 3, 4.5, 7.2 and 9.6 μM (0, 100, 150, 240, 320 ng/μL) gp32. Sample contained ~1 x 10⁵ copies of synthetic HIV-1 RNA; conventional 2-step NASBA included as reference, NTCs as 2-step (no gp32) and 1-step (with 4.5 μM gp32) (b) Comparison of NASBA carried out as conventional 2-step, one step in absence of SSB, and 1-step using 3 μM gp32 for HIV-1 RNA samples ranging from C_t 20 (~1.5 x 10⁶ copies) to C_t 34 (~1.5 x 10² copies).