Propidium monoazide real-time loop-mediated isothermal amplification for specific visualization of viableSalmonellain food

Abstract

Food-borne illnesses caused by Salmonella spp. threaten public health and food safety. Therefore, it is important to develop a rapid and accurate monitoring method to detect viable Salmonella in food. In this study, we developed a calcein-dyed visual real-time loop-mediated isothermal amplification with propidium monoazide treatment (PMA-LAMP) in accordance with the above requirements. The method described here could be a valuable tool for routine screening of viable Salmonella in food.

Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow Immunoassay for Rapid Detection of Salmonella in Food

“…To date, there are more than 80 million cases of foodborne salmonellosis worldwide [5]. The World Health Organization lists Salmonella as a moderately to seriously hazardous foodborne pathogen [6]. Salmonella is found in a wide variety of food products, including poultry, vegetables, eggs, and soybean products, and contamination is common in retail meats [7,8].…”

One Real-Time Fluorescent Loop-Mediated Isothermal Amplification Combined with Propidium Monoazide for Detection of Viable Vibrio Parahaemolyticus in Seafood

“…The proper concentration of PMA working concentration and the maximum treatable concentration of dead V. parahaemolyticus cells PMA, a kind of DNA binding dye, was applied prior to the DNA amplification assay to differentiate viable cells form dead cells by LED light treatment. The proper concentration is important that not only the amplification of dead cell DNA cannot be completely suppressed, but also had no toxicity to viable cells (Zhi et al, 2017;Fang et al, 2018). To obtain the proper concentration of PMA, an amount of 4.5×10 5 CFU/mL heat-killed V. parahaemolyticus dead cells or viable cells were treated with different concentration of PMA from 0 to 80 µM (Fig.…”

“…However, due to the need for complex instruments, complex laboratory procedures, expensive reagents and trained personnel, this method may not be suitable for use in developing countries or in the field (Chen and Ge, 2010;Li et al, 2017). Therefore, an ever-expanding array of novel nucleic acid amplification techniques have been developed to meet the challenge of performing diagnostics without well-equipped equipment in lowresource environments (Fang et al, 2018). On this basis, a variety of nucleic acid detection methods were developed under isothermal conditions.…”

“…The DNA template of Gram-negative bacteria, including V. parahaemolyticus was prepared by the method described previously (Zhi et al, 2017;Fang et al, 2018). The bacteria cells were collected by centrifugation at 10000× g for 5 min.…”

“…To obtain the optimal concentration of PMA treatment, dead cells of V. parahaemolyticus ATCC 17082 were prepared by boiling at 95 °C for 15 min according to the method described previously (Zhi et al, 2017;Fang et al, 2018). To confirm all the bacteria were killed, the cells after treated were cultured on TCBS at 37 °C for 48 h. Then 20 mM PMA (Biotium Inc., Hayward, CA, USA) was added at final concentration of 0, 5, 10, 15, 20, 40 and 80 µM to 500 µL suspension containing 4.5×10 CFU/mL dead V. parahaemolyticus ATCC 17082 in 1.5 mL eppendorf tubes and solution was mixed up and down for several times to make the sample fully in contact with the PMA dye and left in the dark for 5 min.…”