Development of phage immuno-loop-mediated isothermal amplification assays for organophosphorus pesticides in agro-products

Abstract

Two immuno-loop-mediated isothermal amplification assays (iLAMP) were developed by using a phage-borne peptide that was isolated from a cyclic eight-peptide phage library. One assay was used to screen eight organophosphorus (OP) pesticides with limits of detection (LOD) between 2 and 128 ng mL(-1). The iLAMP consisted of the competitive immuno-reaction coupled to the LAMP reaction for detection. This method provides positive results in the visual color of violet, while a negative response results in a sky blue color; therefore, the iLAMP allows one to rapidly detect analytes in yes or no fashion. We validated the iLAMP by detecting parathion-methyl, parathion, and fenitrothion in Chinese cabbage, apple, and greengrocery, and the detection results were consistent with the enzyme-linked immunosorbent assay (ELISA). In conclusion, the iLAMP is a simple, rapid, sensitive, and economical method for detecting OP pesticide residues in agro-products with no instrumental requirement.

Figure 1

Schematic diagram of phage iLAMP.

Figure 2

Screening of positive clones by phage ELISA. Eleven clones out of 20 showed significant signal differences with or without parathion-methyl at 450 nm in three replications.

Figure 3

Design of LAMP primers for detection of C11-2. (A) The nucleotide sequence of C11-2, the color signal region was the nucleotide sequence of the cyclic 8-amino-acid peptide. The sequences used for LAMP primers are indicated by bold lines. (B) Information of the primers, a forward inner primer (FIP) consisted of the complementary sequence of F1 (F1c) and F2, and a backward inner primer (BIP) consisted of B1c and B2. The outer primers F3 and B3 are required for initiation of the LAMP reaction.

Figure 4

Sensitivity and specificity of the LAMP reaction. The sensitivity was evaluated on (A) HNB visualization of color change and (B) 2% agarose gel electrophoresis analysis of the LAMP products. Specificity was assessed by detecting 2 × 10⁶ pfu mL^–1 helper phage M13K07 and 1 mg μL^–1 M13KE vector, the result was observed by (C) color change and (D) 2% agarose gel electrophoresis. M represented 2000 bp DNA mark; 1 represented positive control; 8 and CK represented negative control; 2 to 7 respectively represented 8.5 × 10⁴, 1.7 × 10⁴, 8.5 × 10³, 1.7 × 10³, 8.5 × 10², and 1.7 × 10² pfu mL^–1 C11-2.

Figure 5

ELISA for phage C11-2. The LOD was calculated as the mean of negative OD₄₅₀ (0.137) plus 3 standard deviations (3 × 0.008), which was at 0.161 (n = 3).

Figure 6

Sensitivity of the second iLAMP. Concentration range of parathion-methyl standard assayed by the second iLAMP. (A) HNB-visualized. (B) 2.0% agarose gel electrophoresis, M represented 2000 bp DNA mark, 1 represented negative control, 9 represented positive control, 2 to 8 respectively represented 8, 4, 2, 1, 0.5, 0.25, and 0 ng mL^–1 parathion-methyl. (C) turbidimeter.