Isothermal DNA amplification strategies for duplex microorganism detection

Santiago‐Felipe, Sara; Tortajada-Genaro, Luis Antonio; Morais, Sergi; Puchades, Rosa; Maquieira, Ángel

doi:10.1016/j.foodchem.2014.11.080

Cited by 54 publications

(49 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…For instance, 9% of RPA primers were below 30 bases and 7% were over 35 bases long (36 -45 bases). Short sequences of 20 -23 bases were used in studies for which primers also served as capture probes (11,12 ). Ten percent of primers sequences had a GϩC content Ͻ30% and 3% had a GϩC content Ͼ65%.…”

Section: Reviewmentioning

confidence: 99%

Recombinase Polymerase Amplification for Diagnostic Applications

et al. 2016

View full text Add to dashboard Cite

show abstract

Section: Reviewmentioning

confidence: 99%

Recombinase Polymerase Amplification for Diagnostic Applications

et al. 2016

View full text Add to dashboard Cite

show abstract

“…[16][17][18] and helicase dependent amplification (HDA) 17,19 have all been used for the amplification of DNA in several microfluidic based assays. 22,23 These features make RPA an ideal candidate for rapid and field compatible diagnostic platforms which allow for real-time monitoring of the assay results. 15 Additionally, with LAMP, the design of primers for a specific target is often limited by the need for 4 to 6 suitable primers over a small genomic region and the structure of the amplicon precludes verification of its identity.…”

Section: Introductionmentioning

confidence: 99%

Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform

et al. 2015

View full text Add to dashboard Cite

The widespread dissemination of CTX-M extended spectrum β-lactamases among Escherichia coli bacteria, both in nosocomial and community environments, is a challenge for diagnostic bacteriology laboratories. We describe a rapid and sensitive detection system for analysis of DNA containing the blaCTX-M-15 gene using isothermal DNA amplification by recombinase polymerase amplification (RPA) on a digital microfluidic platform; active matrix electrowetting-on-dielectric (AM-EWOD). The devices have 16,800 electrodes that can be independently controlled to perform multiple and simultaneous droplet operations. The device includes an in-built impedance sensor for real time droplet position and size detection, an on-chip thermistor for temperature sensing and an integrated heater for regulating the droplet temperature. Automatic dispensing of droplets (45 nL) from reservoir electrodes is demonstrated with a coefficient of variation (CV) in volume of approximately 2%. The RPA reaction is monitored in real-time using exonuclease fluorescent probes. Continuous mixing of droplets during DNA amplification significantly improves target DNA detection by at least 100 times compared to a benchtop assay, enabling the detection of target DNA over four-order-of-magnitude with a limit of detection of a single copy within ~15 minutes.

show abstract

“…We believe that this might be due to different enzyme kinetics and experimental protocols for these two assays. While the RPA assay yields double-stranded DNA by Bsu polymerase, the PCR assay uses Taq polymerase to amplify targeted DNA (Piepenburg et al 2006;Santiago-Felipe et al 2015). The PCR assay needs initial temperature denaturation at 95C and temperature cycling between 60 and 95C, whereas the RPA does not require the initial denaturation step or temperature cycling.…”

Section: Discussionmentioning

confidence: 99%

“…Hence, a higher multiplexing ability can be assumed for detecting different targets using the RPA assay. Previous studies showed the practicality of using multiplex applications of RPA for the detection of pathogenic bacteria (Kersting et al 2014;Santiago-Felipe et al 2015).…”

Section: Discussionmentioning

confidence: 99%

Rapid Detection of Salmonella Enterica Serovar Enteritidis from Eggs and Chicken Meat by Real‐Time Recombinase Polymerase Amplification in Comparison with the Two‐Step Real‐Time PCR

Kim

Lee

2016

Journal of Food Safety

View full text Add to dashboard Cite

Salmonella is the most common cause of foodborne outbreaks throughout the world and Salmonella enterica serovar Enteritidis is one of the major causes of salmonellosis. We describe a rapid, sensitive and inexpensive method for the detection of S.Enteritidis from eggs and chicken meat using the real-time recombinase polymerase amplification (Real-time RPA) and compared this assay with the two-step real-time polymerase chain reaction (PCR) (Real-time PCR). The detection sensitivity of the RPA and PCR assays was identical in pure culture, however, the reaction time was as short as 10 min compared with 40 min for the PCR assay. In food application, the detection sensitivity of the RPA assay was 10 cfu/g from eggs and 100 cfu/g from chicken samples without additional DNA purification steps before the amplification -therefore, the sensitivity of the RPA assay was 100 times greater from eggs and 10 times greater from chicken than the PCR assay. PRACTICAL APPLICATIONSNumerous epidemiological investigations attributed the source of sporadic Salmonella outbreaks as poultry and poultry-by-products including eggs. This is the first study in which a real-time RPA was developed to detect the Salmonella Enteritidis specifically. The whole procedure is fairly simple and does not require specific equipment, making it potentially applicable in the monitoring and detection of S. Enteritidis in eggs and poultry products.

show abstract

Isothermal DNA amplification strategies for duplex microorganism detection

Abstract: A valid solution for micro-analytical systems is the selection of a compatible 10

Cited by 54 publications

References 21 publications

Recombinase Polymerase Amplification for Diagnostic Applications

Recombinase Polymerase Amplification for Diagnostic Applications

Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform

Rapid Detection of Salmonella Enterica Serovar Enteritidis from Eggs and Chicken Meat by Real‐Time Recombinase Polymerase Amplification in Comparison with the Two‐Step Real‐Time PCR

Contact Info

Product

Resources

About