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. 2024 Oct 7;13(19):3188.
doi: 10.3390/foods13193188.

Rolling Circle Amplification-Enabled Ultrasensitive Point-of-Care Test Method for Aflatoxin B1 in the Environment and Food

Hongyu Duan  1 , Yuan Zhao  1 , Xiaofeng Hu  1 , Meijuan Liang  1 , Xianglong Yang  1 , Li Yu  1 , Behrouz Tajdar Oranj  2 , Valentin Romanovski  3 , Peiwu Li  1 , Zhaowei Zhang  1
Affiliations

Rolling Circle Amplification-Enabled Ultrasensitive Point-of-Care Test Method for Aflatoxin B1 in the Environment and Food

Hongyu Duan et al. Foods. .

Abstract

Aflatoxin B1 (AFB1) contamination poses a fatal risk to human beings and urgently needs highly sensitive detection for environmental monitoring and food safety. However, the existing challenges are the unsatisfied sensitivity of the immunoassay methods and the complex matrix effect. Rolling circle amplification (RCA) is a promising method for nucleic acid isothermal amplification due to its high specificity and sensitivity. Herein, we constructed a general RCA-based point-of-care test method (RCA-POCT). With biotinylated antibodies, streptavidin, and biotinylated RCA primers, we realized the signal transduction and preliminary signal amplification. In this way, the fluorescent signal of the immunocomplex on the microwells was greatly enhanced. Under optimal conditions, we recorded sensitive detection limits for aflatoxin B1 (AFB1) of 1.94, 16.3, and 37.7 fg/mL (femtogram per microliter), and wide linear ranges with 5 × 10-6 to 5, 5 × 10-5 to 5, and 5 × 10-5 to 5 ng/mL in the irrigation water, field soil, and peanut samples, respectively. Satisfactory recovery, specificity, repeatability, and reproducibility were observed. The RCA-POCT was validated by comparing it to the HPLC method. This work provides a general RCA-assisted detection method for AFB1 in the environment and food.

Keywords: aflatoxin; environmental monitoring; food safety; rolling circle amplification.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Schematic diagram of the principle of the RCA−POCT.
Figure 2
Figure 2
Polyacrylamide gel electrophoresis of the DNA molecules and products. (M: DNA ladder; Lane 1—primer; Lane 2—padlock; Lane 3—circular DNA; Lane 4—the hybridization of primer and circular DNA; Lanes 5–7—RCA products with 1 h, 2 h, and 3 h amplification time).
Figure 3
Figure 3
Optimization of RCA. (A) Influence of the concentration of streptavidin (0, 1, 5, 10, 15, and 20 µg/mL). (B) Influence of the concentration of primer (0, 0.48, 0.72, 0.96, 1.2, and 1.44 µM). (C) Influence of the concentration of circular DNA (0, 0.3, 0.6, 0.9, 1.2, and 1.5 µM). (D) Influence of the concentration of phi29 DNA polymerase (0, 0.1, 0.2, 0.3, 0.4, and 0.5 units/μL). (E) Influence of the RCA reaction time (0, 1.5, 2.0, 2.5, 3.0, and 3.5 h). (F) Influence of the concentration of signal probe (0, 0.1, 0.5, 1.0, 1.5, and 2.0 µM).
Figure 4
Figure 4
The detection of AFB1 with the RCA−POCT. (A) The calibration curves of AFB1 in irrigation water samples. (B) Selectivity tests for the detection of AFB1 over other toxins. (C) Correlation between AFB1 analysis data from 36 samples using HPLC (x−axis) and the RCA−POCT (y−axis).
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