Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review

Craw, Pascal; Balachandran, W.

doi:10.1039/c2lc40100b

Cited by 650 publications

(526 citation statements)

References 169 publications

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“…[8][9][10][11][12][13][14][15][16][17][50][51][52][53][54][55] Reliable quantification of mRNA and microRNA levels for diagnostic and prognostic purposes requires optimal RNA quality and quantity. However, the different instances along the RNA and miRNA analysis workflow can lead to length, sequence, and structure-specific biases in the final expression analysis of a given set of small RNAs.…”

Section: Challenges Faced By the Main Technologies In Nucleic Acmentioning

confidence: 99%

“…Equipment and analysis costs, throughput, and normalization 6,7 still represent obstacles for large-scale nucleic acid biomarker validation [8][9][10][11][12][13][14] and adoption in the clinics [15][16][17] Differences in the nucleic acid purification method, detection technology, and laboratory protocol often lead to different profiling results. Consequently, interpretation of differential expression data and comparisons across different studies has to be done with a note of caution, as standard deviations can span several orders of magnitude.…”

Section: Introductionmentioning

confidence: 99%

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Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine

et al. 2016

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Nucleic acid biomarkers have enormous potential in non-invasive diagnostics and disease management. In medical research and in the near future in the clinics, there is a great demand for accurate miRNA, mRNA, and ctDNA identification and profiling. They may lead to screening of early stage cancer that is not detectable by tissue biopsy or imaging. Moreover, because their cost is low and they are non-invasive, they can become a regular screening test during annual checkups or allow a dynamic treatment program that adjusts its drug and dosage frequently. We briefly review a few existing viral and endogenous RNA assays that have been approved by the Federal Drug Administration. These tests are based on the main nucleic acid detection technologies, namely, quantitative reverse transcription polymerase chain reaction (PCR), microarrays, and next-generation sequencing. Several of the challenges that these three technologies still face regarding the quantitative measurement of a panel of nucleic acids are outlined. Finally, we review a cluster of microfluidic technologies from our group with potential for point-of-care nucleic acid quantification without nucleic acid amplification, designed to overcome specific limitations of current technologies. We suggest that integration of these technologies in a modular design can offer a low-cost, robust, and yet sensitive/selective platform for a variety of precision medicine applications.

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Section: Challenges Faced By the Main Technologies In Nucleic Acmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine

et al. 2016

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“…The multifunctional microfluidic system could detect a few microorganisms quantitatively and simultaneously. Also, they designed different aptamer-and immuno-NASBA assays, which can be used to monitor molecular profiling in serum samples [85] or detect waterborne pathogens [38].…”

Section: Nasba: Nucleic Acid Sequence-based Amplificationmentioning

confidence: 99%

“…This review will discuss different isothermal amplification methods for nucleic acids and their integration into miniaturized systems. Some already published reviews gave a broad overview of that field [36][37][38][39], however, we will have a special view on the most recent strategies to amplify and quantify target molecules in samples. Since the focus of this article is the integration of these technologies into miniaturized systems, the molecular background of the isothermal methods is just briefly described.…”

Section: Introductionmentioning

confidence: 99%

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Tröger¹,

Niemann²,

Gärtig³

et al. 2015

J Nanomed Nanotechnol

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Nucleic acid amplification technologies (NAATs) offer the most sensitive tests in the clinical laboratory. These techniques are used as a powerful tool for screening and diagnosis of infectious diseases. Isothermal methods, as an alternative to polymerase chain reaction (PCR), require no thermocycling machine and can mostly be performed with reduced time, high throughput, and accurate and reliable results.However, current molecular diagnostic approaches generally need manual analysis by qualified and experienced personal which is a highly complex, time-consuming and labor-intensive task. Thus, the demand for simpler, miniaturized systems and assays for pathogen detection is steadily increasing. Microfluidic platforms and lab-on-a-chip devices have many advantages such as small sample volume, portability and rapid detection time and enable point-of-care diagnosis.In this article, we review several isothermal amplification methods and their implementation in microsystems in relation to quantification of nucleic acids. miniaturized systems can be diverse. The majority of publications are focussed on clinical diagnostics including foodborne organisms for environmental monitoring [10][11][12]. In case of an infectious disease, cultivation and phenotypic characterization are still the standard methods of microbiologists which need days up to weeks to obtain a diagnostic result. Hence, scientists started to make use of nucleic acid amplification methods to accelerate the analysis. The most widespread and well known technique for this purpose is the polymerase chain reaction (PCR) [10,13], which exploits the activity of the DNA polymerase. The method is based on a thermal cycling process consisting of repeated cycles of heating and cooling steps allowing for DNA melting, sequence specific primer binding and enzymatic amplification of the target nucleic acid. The quantitative assessment of target copies is possible by using a real-time PCR approach, where a concentration dependent signal (e.g. fluorescence) increases over time [14]. The latter enables the user to easily assess the pathogen load of patient samples [15][16][17][18]. Since the first example of a PCR on a miniaturized system in 1994 [19,20], numerous devices were presented, which integrate not just the amplification, but also sample preparation and detection steps [9,[21][22][23][24][25][26][27][28]. Just a few of those microsystems have reached the market so far, but some have shown a fascinating potential to replace the classical laboratory-oriented state-of-the art [29][30][31][32][33]. setups has been shown to obtain a similar result [34]. A smaller heat capacity allows for rapid changes in temperature beneficial for the PCR time as well as a higher parallelism of multiple genetic samples [34].However, a major drawback of the integration of PCR to microsystems is the necessity of sophisticated instrumentation. The reaction requires a thermal cycling instrumentation, space and considerable expertise [35]. Therefore, isothermal reactions for the ta...

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“…[12][13][14][15][16] Among these isothermal amplification platforms, recombinase polymerase amplification (RPA) has a number of advantages. The RPA can be performed at body temperature, theoretically alleviating the need for external heating equipment if body heat were to be harnessed to incubate reactions.…”

Section: Introductionmentioning

confidence: 99%

Recombinase Polymerase Amplification-Based Assay to Diagnose Giardia in Stool Samples

Crannell

Cabada

Castellanos-González

et al. 2015

The American Society of Tropical Medicine and Hygiene

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Abstract. Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance.

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Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review

Cited by 650 publications

References 169 publications

Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine

Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Recombinase Polymerase Amplification-Based Assay to Diagnose Giardia in Stool Samples

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