Nucleic Acid Isothermal Amplification Technologies—A Review

Real Time RT-PCR developed in recent years represents an useful tool in the diagnosis of RNA viruses. In order to accurately quantify and normalize a RNA target, efficiency of reverse-transcription must be considered. In this study a cRNA-standard based quantitative Real Time RT-PCR have been developed for HRV quantification on bronchoalveolar lavage (BAL) specimens. Results has been compared to a quantitative plasmid standard-based Real Time RT-PCR previously developed by us.Large amount of pHRV was linearized and purified. Blunt ends were generated and cRNA production was carried out. Dilutions of cRNA were generated and dynamic range, intraand inter-test variability, sensitivity and limit of detection were evaluated. Sixty-seven BAL, previously resulted positive to our plasmid standard-based method, were evaluated using cRNA-standard quantification. cRNA curve showed a broad dynamic range with a good intra-and inter-test variability, with an average of 3.23 threshold cycles more in comparison to plasmid standard-based curve. In terms of specimen quantification, a difference of 1.07log was found, showing a significant underrate using plasmid standard-based quantification.The method for cRNA-standard construction seems more suitable for quantification of RNA viruses, in order to normalize the quantification in reverse-transcription.

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“…Among these methods, Real Time RT-PCR is the most used and many platforms are available in the routine laboratories (1,2,3).…”

Section: Introductionmentioning

confidence: 99%

Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR

et al. 2010

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“…The simple reaction scheme is one of the major advantages of HDA over other methods that often include more than one set of primers and just a singular enzyme [39]. There is no need for an initial denaturation step for DNA and the reaction has the ability to detect RNA [108][109][110][111].…”

Section: E Colimentioning

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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“…However, new PCR technologies, such as isothermal PCR, are of particularly practical use in the diagnosis of enteric fever, as these methods allow for the possibility of developing less-complicated and less-expensive machinery than is necessary for conventional PCR. Several isothermal PCR technologies have been developed (Gill & Ghaemi, 2008), including strand displacement amplification (SDA) (Walker et al, 1992), loop-mediated amplification (LAMP) (Notomi et al, 2000), and helicase-dependent amplification (HDA) (Vincent et al, 2004). Recently, Francois et al have examined the robustness of LAMP for bacterial diagnostic applications using S. Typhi as the target organism (Francois et al, 2011), and demonstrated that LAMP is more sensitive than conventional qPCR and is also a very robust, innovative and powerful molecular diagnostic method.…”

Section: Future Perspectivesmentioning

confidence: 99%