Simplifying Nucleic Acid Amplification from Whole Blood with Direct Polymerase Chain Reaction on Chitosan Microparticles

Tremendous advances have been made in the development of portable nucleic acid amplification devices for near-patient use. However, the true limitation in the realization of nucleic acid amplification tests (NAATs) for near-patient applications is not the amplification reaction, it is the complexity of the sample preparation. Conventional approaches require several precise intervention steps during the protocol. There are numerous reports in the literature that mimic the sample preparation procedure within a lab-on-a-chip device or cartridge, but these systems require a high number of integrated steps, making the devices and/or their supporting equipment too complex to meet the necessary cost targets and regulatory requirements for near-patient applications. Here we report a simplified method to purify and amplify DNA from complex samples in a minimal number of steps. We show that chitosan-coated microparticles can lyse human cells and capture the released DNA in a single mechanical agitation step, and we show that bound DNA can be amplified directly from the microparticle surface when the magnetic microparticles are transferred to a polymerase chain reaction (PCR). This procedure eliminates (i) the use of PCR-inhibiting reagents (e.g., chaotropic salts and alcohol) and (ii) the washing and elution steps that are required to remove these reagents and release DNA in typical NAAT sample preparation methods. To illustrate the use of this direct PCR method in diagnostics, we amplify human genomic DNA sequences from a ∼1 μL droplet of whole blood, and we amplify plasmid DNA spiked into whole blood droplets to represent circulating viral DNA or cell-free DNA. The qPCR threshold cycle for direct PCR from whole blood is comparable to that of direct PCR with purified DNA, demonstrating that the lysis and capture steps effectively bind DNA and sufficiently enable its amplification. Furthermore, the efficient amplification of plasmid DNA spiked into whole blood proves that the large mass of human genomic DNA captured from the lysed cells does not inhibit the capture and amplification of other circulating DNA. We anticipate that this new streamlined method for preparing DNA for amplification will expand the diagnostic applications of nucleic acid amplification tests, in particular for near-patient applications

Effects of CMV as GM on the NO₃^- and NH₄⁺ contents in paddy soil at maturity stage of rice plants.

<p>Bars indicate standard error (n = 3). Different letters indicate significantly different means at <i>P</i> < 0.05.</p

Effect of CMV as GM on rice grain yield, GWP and GHGI of N₂O from monocropped rice field.

<p>Effect of CMV as GM on rice grain yield, GWP and GHGI of N₂O from monocropped rice field.</p

Effect of CMV as GM on the populations of nitrifying and denitrifying bacteria in paddy soil at maturity stage of rice plants.

<p>Bars indicate standard error (n = 3). Different letters indicate significantly different means at <i>P</i> < 0.05.</p

The effects of CMV as GM on cumulative amount and emission factor of N₂O in the monocropped rice system during the growing period of rice plants.

<p>The effects of CMV as GM on cumulative amount and emission factor of N₂O in the monocropped rice system during the growing period of rice plants.</p

The N₂O emission fluxes during the growing period of rice plants.

<p>TP was the time of transplanting rice seedlings, and LSA was the time of 20 days before rice harvest. Bars indicate standard error (n = 3).</p

Effects of CMV as GM on the activities of nitrate and nitrite reductase in paddy soil at maturity stage of rice plants.

<p>Bars indicate standard error (n = 3). Different letters indicate significantly different means at <i>P</i> < 0.05.</p

Effects of TiO₂ nanoparticles on wheat (<i>Triticum aestivum L</i>.) seedlings cultivated under super-elevated and normal CO₂ conditions

<div><p>Concerns over the potential risks of nanomaterials to ecosystem have been raised, as it is highly possible that nanomaterials could be released to the environment and result in adverse effects on living organisms. Carbon dioxide (CO₂) is one of the main greenhouse gases. The level of CO₂ keeps increasing and subsequently causes a series of environmental problems, especially for agricultural crops. In the present study, we investigated the effects of TiO₂ NPs on wheat seedlings cultivated under super-elevated CO₂ conditions (5000 mg/L CO₂) and under normal CO₂ conditions (400 mg/L CO₂). Compared to the normal CO₂ condition, wheat grown under the elevated CO₂ condition showed increases of root biomass and large numbers of lateral roots. Under both CO₂ cultivation conditions, the abscisic acid (ABA) content in wheat seedlings increased with increasing concentrations of TiO₂ NPs. The indolepropioponic acid (IPA) and jasmonic acid (JA) content notably decreased in plants grown under super-elevated CO₂ conditions, while the JA content increased with increasing concentrations of TiO₂ NPs. Ti accumulation showed a dose-response manner in both wheat shoots and roots as TiO₂ NPs concentrations increased. Additionally, the presence of elevated CO₂ significantly promoted Ti accumulation and translocation in wheat treated with certain concentrations of TiO₂ NPs. This study will be of benefit to the understanding of the joint effects and physiological mechanism of high-CO₂ and nanoparticle to terrestrial plants.</p></div

Physiological responses of wheat seedlings upon exposure to different levels of CO₂.

<p>Values are presented as mean±SD, error bars represent standard deviation (sample size, n = 64 under super-elevated CO₂ condition and n = 48 under normal CO₂ condition). Lower letters represent significant difference at p<0.05 between super-elevated and normal CO₂ treatments.</p

TEM image and particle size distribution of TiO₂ NPs.

<p>TEM image and particle size distribution of TiO₂ NPs.</p