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

Chinese Milk Vetch as Green Manure Mitigates Nitrous Oxide Emission from Monocropped Rice System in South China - Fig 1

The mean monthly temperature (a) and total monthly precipitation (b) at the experimental site.</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

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

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

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

Capture and Direct Amplification of DNA on Chitosan Microparticles in a Single PCR-Optimal Solution

While nucleic acid amplification tests have great potential as tools for rapid diagnostics, complicated sample preparation requirements inhibit their use in near-patient diagnostics and low-resource-setting applications. Recent advancements in nucleic acid purification have leveraged pH-modulated charge switching polymers to reduce the number of steps required for sample preparation. The polycation chitosan (p<i>K</i>_a 6.4) has been used to efficiently purify DNA by binding nucleic acids in acidic buffers and then eluting them at a pH higher than 8.0. Though it is an improvement over conventional methods, this multistep procedure has not transformed the application of nucleic acid amplification assays. Here we describe a simpler approach using magnetic chitosan microparticles that interact with DNA in a manner that has not been reported before. The microparticles capture DNA at a pH optimal for PCR (8.5) just as efficiently as at low pH. Importantly, the captured DNA is still accessible by polymerase, enabling direct amplification from the microparticles. We demonstrate quantitative PCR from DNA captured on the microparticles, thus eliminating nearly all of the sample preparation steps. We anticipate that this new streamlined method for preparing DNA for amplification will greatly expand the diagnostic applications of nucleic acid amplification tests

Carbonic Anhydrase IX Targeting Mn(II)-Based Magnetic Resonance Molecular Imaging Probe for Hypoxia Tumors

Physiological hypoxic conditions in the tumor microenvironment and consequential overexpression of carbonic anhydrase IX (CA IX) are two characteristics shared by numerous types of solid malignant tumors. Early detection with hypoxia assessment is crucial to improve the prognosis and therapy outcomes of hypoxia tumors. Herein, using acetazolamide (AZA) as a CA IX-targeting moiety, we design and synthesize an Mn(II)-based MR imaging probe (named AZA-TA-Mn) incorporating AZA and two Mn(II) chelates of Mn-TyEDTA on a rigid triazine (TA) scaffold. The per Mn relaxivity of AZA-TA-Mn is 2-fold higher than its monomeric Mn-TyEDTA, which allows it for low-dose imaging of hypoxic tumors. In a xenograft mice model of esophageal squamous cell carcinoma (ESCC), a low dosage of AZA-TA-Mn (0.05 mmol/kg) can selectively produce prolonged and stronger contrast enhancement in the tumor compared to the non-specific Gd-DTPA (0.1 mmol/kg). A competition study of co-injection of free AZA and Mn(II) probes confirms the in vivo tumor selectivity of AZA-TA-Mn, resulting in a more than 2.5-fold decreased tumor-to-muscle contrast-to-noise ratio (ΔCNR) at 60 min post-injection. MR imaging results were further supported by the quantitative analysis of Mn tissue levels, as the co-injection of free AZA resulted in significantly reduced Mn accumulation in tumor tissues. Finally, immunofluorescence staining of tissue sections confirms the positive correlation between the tumor accumulation of AZA-TA-Mn and CA IX overexpression. Hence, using CA IX as the hypoxia biomarker, our results illustrate a practical strategy for the development of novel imaging probes for hypoxic tumors