Closed-tube saltatory rolling circle amplification assay for rapid and visual detection of allergenic cashew nut in foods

Cashew nut allergy shows an increasing prevalence along with its high consumption. Nowadays, food allergy still has no cure, and allergen avoidance is still the standard management, urging a sensitive and convenient cashew nut detection method. Herein, saltatory rolling circle amplification (SRCA), loop-mediated isothermal amplification (LAMP), as well as polymerase chain reaction (PCR) targeting the Ana o 2 gene were proposed and compared. In addition, a closed-tube SRCA assay using hydroxynaphthol blue (HNB) as a color indicator (HNBSRCA) was developed to realize visual on-site detection of cashew nut. SRCA and HNB-SRCA can detect 75.3 fg of cashew nut DNA, which is 10- and 100-times more sensitive than LAMP and PCR, respectively. SRCA and HNBSRCA exhibited a relative sensitivity of 0.01% in a binary mixture, which is comparable to LAMP and 10-times higher than that of PCR. When combined with a rapid DNA extraction method, SRCA and HNB-SRCA performed better applicability compared to LAMP and PCR. Additionally, compared to SRCA using electrophoresis to analyze the amplification products, the visual closed-tube HNB-SRCA combined with the rapid DNA extraction protocol is a better potential cashew nut on-site detection strategy in foods.