Loop-mediated isothermal amplification for visual detection of Vibrio parahaemolyticus using gold nanoparticles.

ABSTRACT

Loop-mediated isothermal amplification (LAMP) eradicates the need of thermocycler in DNA amplification. Signals are usually obtained via fluorometry or turbidimetry, but such methods need improvement in order to become more effortless and reliable. The authors describe a set of six specific primers targeting the species-specific tlh gene of Vibrio parahaemolyticus which were used in accelerated LAMP reaction. Gold nanoparticles (AuNPs) were functionalized with streptavidin (Avidin-AuNPs), and engineered to signal the LAMP reaction. Two of the loop primers for LAMP were biotinylated and then can produce a DNA that can cause clusterization of Avidin-AuNPs based on the formation of avidin-biotin complex. This leads to a color change of the solution from red to blue. Amplification is completed within 30 min and can be visually detected within 5 min. The detection limit of the method is found to be 8.6 cfu per reaction. This visual detection scheme does not require any fluorescent reagents and detection instruments. Conceivably, the method has a wide scope because such Avidin-AuNPs can be used as nanoprobes for a variety of other LAMP products. This rapid and universal strategy holds promise in point of care testing and food testing, particularly in resource-limited regions. Graphical abstract Six specific primers (two of them are biotinylated) were used to realize the accelerated Loop-Mediated Isothermal Amplification. Streptavidin modified gold nanoparticles (Avidin-AuNPs) cluster on the DNA products, leading to the apparent change of color from red to blue, which is readily identified even by unaided eye.