Comparison of isothermal helicase-dependent amplification and PCR for the detection of Mycobacterium tuberculosis by an electrochemical genomagnetic assay

Barreda-García, Susana; Miranda‐Castro, Rebeca; de‐los‐Santos‐Álvarez, Noemí; Miranda‐Ordieres, Arturo J.; Lobo-Castañón, Marı́a Jesús

doi:10.1007/s00216-016-9514-z

Cited by 25 publications

(20 citation statements)

References 27 publications

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“…The application of a fine-tuned primer set for isothermal DNA amplification, helicase-dependent amplification (HDA) or recombinase polymerase amplification (RPA), to PCR has reasonable chance of succeeding [17]. Instead, the opposite situation in which primers designed for PCR are used for amplification at a constant temperature, although possible, could be more demanding.…”

Section: Resultsmentioning

confidence: 99%

A Quantitative PCR-Electrochemical Genosensor Test for the Screening of Biotech Crops

Moura-Melo

Miranda‐Castro

de‐los‐Santos‐Álvarez

et al. 2017

Sensors

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The design of screening methods for the detection of genetically modified organisms (GMOs) in food would improve the efficiency in their control. We report here a PCR amplification method combined with a sequence-specific electrochemical genosensor for the quantification of a DNA sequence characteristic of the 35S promoter derived from the cauliflower mosaic virus (CaMV). Specifically, we employ a genosensor constructed by chemisorption of a thiolated capture probe and p-aminothiophenol gold surfaces to entrap on the sensing layer the unpurified PCR amplicons, together with a signaling probe labeled with fluorescein. The proposed test allows for the determination of a transgene copy number in both hemizygous (maize MON810 trait) and homozygous (soybean GTS40-3-2) transformed plants, and exhibits a limit of quantification of at least 0.25% for both kinds of GMO lines.

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Section: Resultsmentioning

confidence: 99%

A Quantitative PCR-Electrochemical Genosensor Test for the Screening of Biotech Crops

Moura-Melo

Miranda‐Castro

de‐los‐Santos‐Álvarez

et al. 2017

Sensors

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“…Ru(NH 3 ) 6 2+ ), 37 particles 34 to amplify the read out. [38][39][40] The literature is rather rich ( Table 1) and some examples will be highlighted in the following section. Electrochemical detection of E. coli and S. aureus using a LAMP amplicon was demonstrated by Ahmed et al 37 Abdalhai et al reported an electrochemical genosensor for the detection of pathogenic E. coli O157:H7 in fresh beef samples.…”

Section: Imprintingmentioning

confidence: 99%

Electrochemical Methodologies for the Detection of Pathogens

et al. 2018

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Bacterial infections remain one of the principal causes of morbidity and mortality worldwide. The number of deaths due to infections is declining every year by only 1% with a forecast of 13 million deaths in 2050. Among the 1400 recognized human pathogens, the majority of infectious diseases is caused by just a few, about 20 pathogens only. While the development of vaccinations and novel antibacterial drugs and treatments are at the forefront of research, and strongly financially supported by policy makers, another manner to limit and control infectious outbreaks is targeting the development and implementation of early warning systems, which indicate qualitatively and quantitatively the presence of a pathogen. As toxin contaminated food and drink are a potential threat to human health and consequently have a significant socioeconomic impact worldwide, the detection of pathogenic bacteria remains not only a big scientific challenge but also a practical problem of enormous significance. Numerous analytical methods, including conventional culturing and staining techniques as well as molecular methods based on polymerase chain reaction amplification and immunological assays, have emerged over the years and are used to identify and quantify pathogenic agents. While being highly sensitive in most cases, these approaches are highly time, labor, and cost consuming, requiring trained personnel to perform the frequently complex assays. A great challenge in this field is therefore to develop rapid, sensitive, specific, and if possible miniaturized devices to validate the presence of pathogens in cost and time efficient manners. Electrochemical sensors are well accepted powerful tools for the detection of disease-related biomarkers and environmental and organic hazards. They have also found widespread interest in the last years for the detection of waterborne and foodborne pathogens due to their label free character and high sensitivity. This Review is focused on the current electrochemical-based microorganism recognition approaches and putting them into context of other sensing devices for pathogens such as culturing the microorganism on agar plates and the polymer chain reaction (PCR) method, able to identify the DNA of the microorganism. Recent breakthroughs will be highlighted, including the utilization of microfluidic devices and immunomagnetic separation for multiple pathogen analysis in a single device. We will conclude with some perspectives and outlooks to better understand shortcomings. Indeed, there is currently no adequate solution that allows the selective and sensitive binding to a specific microorganism, that is fast in detection and screening, cheap to implement, and able to be conceptualized for a wide range of biologically relevant targets.

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“…Recently, a novel isothermal real-time detection method (HDA-TaqMan) that combines the advantages of both HDA and a TaqMan assay was reported for detection of biothreat organisms: Vibrio cholerae and Bacillus anthracis. In this technique, the reactions of DNA unwinding, primer annealing, polymerization, probe hybridization, and subsequent hydrolysis by the polymerase are coordinated and synchronized to perform at a single temperature (Barreda-García et al, 2016).…”

Section: Isothermal Gene Amplification Assaysmentioning

confidence: 99%