Correction: Nucleic acid purification from plants, animals and microbes in under 30 seconds.

[This corrects the article DOI: 10.1371/journal.pbio.2003916.].

Nucleic acid purification from plants, animals and microbes in under 30 seconds.

Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries.

Isothermal Point Mutation Detection: Toward a First-Pass Screening Strategy for Multidrug-Resistant Tuberculosis.

Point mutations in DNA are useful biomarkers that can provide critical classification of disease for accurate diagnosis and to inform clinical decisions. Conventional approaches to detect point mutations are usually based on technologies such as real-time polymerase chain reaction (PCR) or DNA sequencing, which are typically slow and require expensive lab-based equipment. While rapid isothermal strategies such as recombinase polymerase amplification (RPA) have been proposed, they tend to suffer from poor specificity in discriminating point mutations. Herein, we describe a novel strategy that enabled exquisite point mutation discrimination with isothermal DNA amplification, using mismatched primers in conjunction with a two-round enrichment process. As a proof of concept, the method was applied to the rapid and specific identification of drug-resistant Mycobacterium tuberculosis using RPA under specific conditions. The assay requires just picogram levels of genomic DNA input, is sensitive and specific enough to detect 10% point mutation loading, and can discriminate between closely related mutant variants within 30 min. The assay was subsequently adapted onto a low-cost 3D-printed isothermal device with real-time analysis capabilities to demonstrate a potential point-of-care application. Finally, the generic applicability of the strategy was shown by detecting three other clinically important cancer-associated point mutations. We believe that our assay shows potential in a broad range of healthcare screening processes for detecting and categorizing disease phenotypes at the point of care, thus reducing unnecessary therapy and cost in these contexts.

Field Demonstration of a Multiplexed Point-of-Care Diagnostic Platform for Plant Pathogens.

Effective disease management strategies to prevent catastrophic crop losses require rapid, sensitive, and multiplexed detection methods for timely decision making. To address this need, a rapid, highly specific and sensitive point-of-care method for multiplex detection of plant pathogens was developed by taking advantage of surface-enhanced Raman scattering (SERS) labeled nanotags and recombinase polymerase amplification (RPA), which is a rapid isothermal amplification method with high specificity. In this study, three agriculturally important plant pathogens (Botrytis cinerea, Pseudomonas syringae, and Fusarium oxysporum) were used to demonstrate potential translation into the field. The RPA-SERS method was faster, more sensitive than polymerase chain reaction, and could detect as little as 2 copies of B. cinerea DNA. Furthermore, multiplex detection of the three pathogens was demonstrated for complex systems such as the Arabidopsis thaliana plant and commercial tomato crops. To demonstrate the potential for on-site field applications, a rapid single-tube RPA/SERS assay was further developed and successfully performed for a specific target outside of a laboratory setting.

Toward Precision Medicine: A Cancer Molecular Subtyping Nano-Strategy for RNA Biomarkers in Tumor and Urine.

Cancer is a heterogeneous disease which manifests as different molecular subtypes due to the complex nature of tumor initiation, progression, and metastasis. The concept of precision medicine aims to exploit this cancer heterogeneity by incorporating diagnostic technology to characterize each cancer patient's molecular subtype for tailored treatments. To characterize cancer molecular subtypes accurately, a suite of multiplexed bioassays have currently been developed to detect multiple oncogenic biomarkers. Despite the reliability of current multiplexed detection techniques, novel strategies are still needed to resolve limitations such as long assay time, complex protocols, and difficulty in interpreting broad overlapping spectral peaks of conventional fluorescence readouts. Herein a rapid (80 min) multiplexed platform strategy for subtyping prostate cancer tumor and urine samples based on their RNA biomarker profiles is presented. This is achieved by combining rapid multiplexed isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) of target RNA biomarkers with surface-enhanced Raman spectroscopy (SERS) nanotags for "one-pot" readout. This is the first translational application of a RT-RPA/SERS-based platform for multiplexed cancer biomarker detection to address a clinical need. With excellent sensitivity of 200 zmol (100 copies) and specificity, we believed that this platform methodology could be a useful tool for rapid multiplexed subtyping of cancers.

Rapid, Single-Cell Electrochemical Detection of Mycobacterium tuberculosis Using Colloidal Gold Nanoparticles.

Tuberculosis (TB) remains a global health threat, with over a third of the world population suffering from the disease, and 1.5 million deaths due to the disease in 2013 alone. Despite significant advances in TB detection strategies in recent years, a bigger push toward detecting TB in the shortest and easiest way possible at the point-of-care (POC) is still in demand. To this end, we have designed a simple yet rapid and sensitive bioassay that detects Mtb DNA electrochemically using colloidal gold nanoparticles. This assay couples rapid isothermal amplification of target DNA that is specific to Mtb with gold nanoparticle electrochemistry on disposable screen printed carbon electrodes. The assay is capable of detecting a positive differential pulse voltammetry (DPV) response from as low as 1 CFU of Mtb bacilli DNA input material, having shown its exquisite sensitivity over a conventional gel based readout. The translation of our assay onto a portable potentiostat was also demonstrated, with promising results. We believe that our assay has significant potential for translation into broader bioassay applications or development as a POC diagnostic tool.

DNA ligase-based strategy for quantifying heterogeneous DNA methylation without sequencing.

BACKGROUND: DNA methylation is a potential source of disease biomarkers. Typically, methylation levels are measured at individual cytosine/guanine (CpG) sites or over a short region of interest. However, regions of interest often show heterogeneous methylation comprising multiple patterns of methylation (epialleles) on individual DNA strands. Heterogeneous methylation is largely ignored because digital methods are required to deconvolute these usually complex patterns of epialleles. Currently, only single-molecule approaches, such as next generation sequencing (NGS), can provide detailed epiallele information. Because NGS is not yet feasible for routine practice, we developed a single-molecule-like approach, named for epiallele quantification (EpiQ). METHODS: EpiQ uses DNA ligases and the enhanced thermal instability of short (≤19 bases) mismatched DNA probes for the relative quantification of epialleles. The assay was developed using fluorescent detection on a gel and then adapted for electrochemical detection on a microfabricated device. NGS was used to validate the analytical accuracy of EpiQ. RESULTS: In this proof of principle study, EpiQ detected with 90%-95% specificity each of the 8 possible epialleles for a 3-CpG cluster at the promoter region of the CDKN2B (p15) tumor suppressor gene. EpiQ successfully profiled heterogeneous methylation patterns in clinically derived samples, and the results were cross-validated with NGS. CONCLUSIONS: EpiQ is a potential alternative tool for characterizing heterogeneous methylation, thus facilitating its use as a biomarker. EpiQ was developed on a gel-based assay but can also easily be adapted for miniaturized chip-based platforms.

High-speed biosensing strategy for non-invasive profiling of multiple cancer fusion genes in urine.

Aberrant chromosal rearrangements, such as the multiple variants of TMPRSS2:ERG fusion gene mutations in prostate cancer (PCa), are promising diagnostic and prognostic biomarkers due to their specific expression in cancerous tissue only. Additionally, TMPRSS2:ERG variants are detectable in urine to provide non-invasive PCa diagnostic sampling as an attractive surrogate for needle biopsies. Therefore, rapid and simplistic assays for identifying multiple urinary TMPRSS2:ERG variants are potentially useful to aid in early cancer detection, immediate patient risk stratification, and prompt personalized treatment. However, current strategies for simultaneous detection of multiple gene fusions are limited by tedious and prolonged experimental protocols, thus limiting their use as rapid clinical screening tools. Herein, we report a simple and rapid gene fusion strategy which expliots the specificity of DNA ligase and the speed of isothermal amplification to simultaneously detect multiple fusion gene RNAs within a short sample-to-answer timeframe of 60min. The method has a low detection limit of 2 amol (1000 copies), and was successfully applied for non-invasive fusion gene profiling in patient urine samples with subsequent validation by a PCR-based gold standard approach.

Enabling miniaturised personalised diagnostics: from lab-on-a-chip to lab-in-a-drop.

The concept of personalised diagnostics is to direct accurate clinical decisions based on an individual's unique disease molecular profile. Lab-on-a-chip (LOC) systems are prime personalised diagnostics examples which seek to perform an entire sample-to-outcome detection of disease nucleic acid (NA) biomarkers on a single miniaturised platform with minimal user handling. Despite the great potential of LOC devices in providing rapid, portable, and inexpensive personalised diagnosis at the point-of-care (POC), the translation of this technology into widespread use has still been hampered by the need for sophisticated and complex engineering. As an alternative miniaturised diagnostics platform free of precision fabrication, there have been recent developments towards a solution-based lab-in-a-drop (LID) system by which an entire laboratory-based diagnostics workflow could be downscaled and integrated within a singular fluid droplet for POC detection of NA biomarkers. In contrast to existing excellent reviews on miniaturised LOC fabrication and individual steps of NA biomarker sensing, we herein focus on miniaturised solution-based NA biosensing strategies suited for integrated LID personalised diagnostics development. In this review, we first evaluate the three fundamental bioassay steps for miniaturised NA biomarker detection: crude sample preparation, isothermal target amplification, and detection readout of amplicons. Then, we provide insights into research advancements towards a functional LID system which integrates all three of the above-mentioned fundamental steps. Finally, we discuss perspectives and future directions of LID diagnostic platforms in personalised medicine applications.

A nanoplasmonic label-free surface-enhanced Raman scattering strategy for non-invasive cancer genetic subtyping in patient samples.

Simple nucleic acid detection methods could facilitate the progress of disease diagnostics for clinical uses. An attractive strategy is label-free surface-enhanced Raman scattering (SERS) due to its capability of providing structural fingerprinting of analytes that are close to or on nanomaterial surfaces. However, current label-free SERS approaches for DNA/RNA biomarker detection are limited to short and synthetic nucleic acid targets and have not been fully realized in clinical samples due to two possible reasons: (i) low target copies in limited patient samples and (ii) poor capability in identifying specific biomarkers from complex samples. To resolve these limitations and enable label-free SERS for clinical applications, we herein present a novel strategy based on multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) to enrich multiple RNA biomarkers, followed by label-free SERS with multivariate statistical analysis to directly detect, identify and distinguish between these long amplicons (∼200 bp). As a proof-of-concept clinical demonstration, we employed this strategy for non-invasive subtyping of prostate cancer (PCa). In a training cohort of 43 patient urinary samples, we achieved 93.0% specificity, 95.3% sensitivity, and 94.2% accuracy. We believe that our proposed assay could pave the way for simple and direct label-free SERS detection of multiple long nucleic acid sequences in patient samples, and thus facilitate rapid cancer molecular subtyping for personalized therapies.

Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes.

Developing quick and sensitive molecular diagnostics for plant pathogen detection is challenging. Herein, a nanoparticle based electrochemical biosensor was developed for rapid and sensitive detection of plant pathogen DNA on disposable screen-printed carbon electrodes. This 60 min assay relied on the rapid isothermal amplification of target pathogen DNA sequences by recombinase polymerase amplification (RPA) followed by gold nanoparticle-based electrochemical assessment with differential pulse voltammetry (DPV). Our method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. On the basis of the speed, sensitivity, simplicity and portability of the approach, we believe the method has potential as a rapid disease management solution for applications in agriculture diagnostics.

Colorimetric TMPRSS2-ERG Gene Fusion Detection in Prostate Cancer Urinary Samples via Recombinase Polymerase Amplification.

TMPRSS2 (Exon 1)-ERG (Exon 4) is the most frequent gene fusion event in prostate cancer (PC), and is highly PC-specific unlike the current serum prostate specific antigen (PSA) biomarker. However, TMPRSS2-ERG levels are currently measured with quantitative reverse-transcription PCR (RT-qPCR) which is time-consuming and requires costly equipment, thus limiting its use in clinical diagnostics. Herein, we report a novel rapid, cost-efficient and minimal-equipment assay named "FusBLU" for detecting TMPRSS2-ERG gene fusions from urine. TMPRSS2-ERG mRNA was amplified by isothermal reverse transcription-recombinase polymerase amplification (RT-RPA), magnetically-isolated, and detected through horseradish peroxidase (HRP)-catalyzed colorimetric reaction. FusBLU was specific for TMPRSS2-ERG mRNA with a low visual detection limit of 10(5) copies. We also demonstrated assay readout versatility on 3 potentially useful platforms. The colorimetric readout was detectable by naked eye for a quick yes/no evaluation of gene fusion presence. On the other hand, a more quantitative TMPRSS2-ERG detection was achievable by absorbance/electrochemical measurements. FusBLU was successfully applied to 12 urinary samples and results were validated by gold-standard RT-qPCR. We also showed that sediment RNA was likely the main source of TMPRSS2-ERG mRNA in urinary samples. We believe that our assay is a potential clinical screening tool for PC and could also have wide applications for other disease-related fusion genes.

Accurate and sensitive total genomic DNA methylation analysis from sub-nanogram input with embedded SERS nanotags.

Sensitive and accurate total genomic DNA methylation analysis was demonstrated by surface-enhanced Raman scattering (SERS) via embedded internal SERS nanotags. The assay was sensitive to 0.2 ng input DNA while differentiating as low as 6.25% changes in DNA methylation. The method could also successfully differentiate cells before and after de-methylating drug-treatment and between tumor and normal biopsies.

Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction.

The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplification method with high potential for molecular diagnostics due to its isothermal nature and high amplification efficiency. However, the use of EXPAR is limited by the high levels of non-specific amplification. Hence, methods that can improve the specificity of EXPAR are desired to facilitate its widespread adoption in practice. Herein, we proposed a strategy to improve EXPAR performance by using molecular enhancers. Eight small molecules were investigated, including ethylene glycol, propylene glycol, betaine, dimethyl sulfoxide (DMSO), trehalose, tetramethylammonium chloride (TMAC), bovine serum albumin (BSA) and single-stranded binding (SSB) proteins. A combination of kinetic and end-point analysis was adopted to investigate how these molecules affected EXPAR performance. Trehalose, TMAC, BSA and SSB proteins were found to have positive effects on EXPAR with trehalose being able to increase the efficiency of EXPAR. In contrast, TMAC, BSA and SSB proteins were shown to increase the specificity of EXPAR. We applied our findings to demonstrate the combination of trehalose and TMAC could simultaneously improve both the efficiency and specificity of an EXPAR-based miRNA detection method. The information provided in this study may serve as a reference to benefit the wider isothermal amplification community.

Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags.

Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research.

A simple, rapid, low-cost technique for naked-eye detection of urine-isolated TMPRSS2:ERG gene fusion RNA.

The TMPRSS2:ERG gene fusion is one of a series of highly promising prostate cancer (PCa) biomarker alternatives to the controversial serum PSA. Current methods for detecting TMPRSS2:ERG are limited in terms of long processing time, high cost and the need for specialized equipment. Thus, there is an unmet need for less complex, faster, and cheaper methods to enable gene fusion detection in the clinic. We describe herein a simple, rapid and inexpensive assay which combines robust isothermal amplification technique with a novel visualization method for evaluating urinary TMPRSS2:ERG status at less than USD 5 and with minimal equipment. The assay is sensitive, and rapidly detects as low as 10(5) copies of TMPRSS2:ERG transcripts while maintaining high levels of specificity.

Rapid and Sensitive Fusion Gene Detection in Prostate Cancer Urinary Specimens by Label-Free Surface-enhanced Raman Scattering.

Recurrent chromosomal rearrangements such as fusion genes are associated with cancer initiation and progression. Prostate cancer (PCa) is a leading cause of cancer-related deaths in men and the TMPRSS2-ERG gene fusion is a recurrent biomarker in about 50% of all prostate cancers. However, current screening tools for TMPRSS2-ERG are generally confined to research settings and hence, the development of a rapid, sensitive and accurate assay for TMPRSS2-ERG detection may aid in clinical PCa diagnosis and treatment. Herein, we described a new strategy for non-invasive TMPRSS2-ERG detection in patient urinary samples by coupling of isothermal reverse transcription-recombinase polymerization amplification (RT-RPA) to amplify TMPRSS2-ERG transcripts and surface-enhanced Raman scattering (SERS) to directly detect the amplicons. This novel coupling of both techniques allows rapid and quantitative TMPRSS2-ERG detection. Our assay can specifically detect as low as 103 copies input of TMPRSS2-ERG transcripts and was successfully applied to clinical PCa urinary samples. Hence, we believe our assay is a potential clinical screening tool for TMPRSS2-ERG in PCa and may have broad applications in detecting other gene fusion transcripts in other diseases.

Highly sensitive DNA methylation analysis at CpG resolution by surface-enhanced Raman scattering via ligase chain reaction.

Sensitive and accurate DNA methylation analysis at CpG resolution was demonstrated using surface-enhanced Raman scattering (SERS) via ligase chain reaction (LCR). The method was sensitive to 10% changes in methylation and the accuracy of methylation estimates in cells and serum DNA validated with sequencing. The LCR/SERS approach may have broad applications as an alternative (epi)genetic detection method.

A simple bridging flocculation assay for rapid, sensitive and stringent detection of gene specific DNA methylation.

The challenge of bringing DNA methylation biomarkers into clinic is the lack of simple methodologies as most current assays have been developed for research purposes. To address the limitations of current methods, we describe herein a novel methyl-protein domain (MBD) enrichment protocol for simple yet rapid and highly stringent selection of highly methylated DNA from limiting input samples. We then coupled this with a DNA-mediated flocculation assay for rapid and low cost naked-eye binary evaluation of highly methylated genes in cell line and blood DNA. The low resource requirements of our method may enable widespread adoption of DNA methylation-based diagnostics in clinic and may be useful for small-scale research.