Resumen de Recombinase Polymerase Amplification Combined with Lateral Flow Strip for Listeria monocytogenesDetection in Food
Xin‐jun Du, Yu‐xuan Zang, Hai‐bin Liu, Ping Li, Shuo Wang
Listeria monocytogenesis an important food‐borne pathogenic bacterium that causes human disease, resulting in economic losses worldwide. The current detection methods for L. monocytogenesare not well suited for direct field testing because they involve complicated, time‐consuming operations. A simple, efficient method is vital for L. monocytogenesdetection. In this study, we combined isothermal recombinase polymerase amplification (RPA) with a lateral flow (LF) strip to rapidly and reliably detect L. monocytogenes. In the presence of biotin‐ and digoxin‐modified primers, RPA produced numerous digoxin‐ and biotin‐attached duplex DNA products. These products were detected on an LF strip via dual immunoreactions (digoxin on the duplex DNA reacted with the anti‐digoxin antibody on the gold nanoparticle (Au‐NP) and the biotin on the duplex DNA captured by the streptavidin on the LF test zone). The accumulation of Au‐NPs produced characteristic bands, enabling the visual detection of L. monocytogeneswithout instrumentation. This assay could be used to detect L. monocytogeneswithin 15 min, including DNA amplification with RPA for 10 min at 39 °C and visualization of the amplicons by LF strips for 5 min. Experiments confirmed a detection limit as low as 300 fg of DNA and 1.5 × 101CFU in pure cultures. Furthermore, RPA‐LF exhibited no cross‐reactions with pathogens. Evaluation of the method with food samples indicated that the detection limit was substantially improved to 1.5 × 10° CFU for the original bacterial content in 25 g/mL samples after enrichment for 6 hr. RPA‐LF can be used as a sensitive and rapid detection technique for L. monocytogenes. Recombinase polymerase amplification (RPA) can amplify target DNA at 37 to 42 °C without a thermal cycler. Lateral flow (LF) strips are portable, cheap and easy to operate. RPA combined with LF strips to detect Listeria monocytogenescan be widely used in remote areas.
