After incubating for 1 h at 37 C, the plate was washed eight times with PBST and 100 L of parathion-methyl (100 ng mLC1 in PBS) was added for 1 h at 37 C to compete with the binding phage from the coating antibody

After incubating for 1 h at 37 C, the plate was washed eight times with PBST and 100 L of parathion-methyl (100 ng mLC1 in PBS) was added for 1 h at 37 C to compete with the binding phage from the coating antibody. 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 Z-DQMD-FMK residues in agro-products with no instrumental requirement. Introduction Organophosphorus (OP) pesticides are widely used in agriculture for the control of sucking and biting insects, including fruit flies, stem borers, mosquitoes, and cereal bugs. However, OP pesticides are considered hazardous substances because of their toxicity to nontarget species and periodic persistence problems in the environment.1 Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, sensitivity, and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity.2 The LAMP method has three main advantages. First, all reactions can be specifically carried out under isothermal conditions, and do Z-DQMD-FMK not require a denatured DNA template. Compared to conventional PCR and real-time PCR assays, specialized and/or expensive equipment is not necessary, and there are fewer preparation Z-DQMD-FMK steps.3,4 Second, the amplification efficiency is extremely high so that high amounts of amplification products can be obtained.5 Third, LAMP results can be read by the naked eye, on the basis of the change of turbidity.6 Alternatively, the LAMP products can be visualized by small molecules such as fluorescein isothiocyanate (FITC), hydroxynaphthol blue (HNB), and digoxigenin (DIG).7,8 Since the first description of the LAMP method in 2000,9 many LAMP assays have been developed for detection of pathogenic microorganisms,10,11 genetically modified ingredients,12,13 tumor detection,14,15 and embryo sex identification.16,17 However, no investigator has reported a LAMP assay for the detection of pesticides since most pesticides are low-molecular weight chemical compounds and do not contain nucleic acids. That LAMP technology likely was overlooked in the environmental field should be quite a concern. Since phage peptide display was first reported,18 it has been a powerful tool for a variety of applications, including the isolation of peptide ligands for antibodies and enzymes,19?21 antibody engineering,22,23 and the isolation of receptor peptides for small molecules.24,25 A phage display random peptide library, which displays extensive random peptides on the N terminus of the minor coat protein g3p of the filamentous phage M13, can be used for this purpose. Peptides with specific affinities or activities toward targets can be screened from the peptide library.26?28 Phage g3p-displayed Rabbit Polyclonal to AP-2 short peptide libraries have been approved to be efficient tools for selecting mimotope Z-DQMD-FMK peptides of an array of compounds, including metabolites of pyrethroid insecticides,29 deoxynivalenol,30 zearalenone,31 ochratoxin A (OTA),32 and aflatoxin.33,34 The unique characteristics of a phage-borne peptide that connects the peptide with affinity to a target on the phage particles containing nucleic Z-DQMD-FMK acids (single-stranded DNA) encoding the peptide make them excellent reagents to develop LAMP assays for small molecules. To test this feasibility, we isolated four phage-borne peptides with specific affinities to a monoclonal antibody (mAb) against OP pesticides. This study describes our systemic approach to the development of the iLAMP using one phage-borne peptide with the highest sensitivity and validating the assay with application to several agricultural samples. Materials and Methods Reagents All reagents were of analytical grade unless specified otherwise. Parathion-methyl, chlorpyrifos-methyl, azinphos-methyl, dimethoate, fenitrooxon, EPN, paraoxon-ethyl, paraoxon-methyl, dicapthon, cyanophos, and famphur were all purchased from Dr. Ehrenstorfer (Germany). Other pesticide standards were provided by the Jiangsu Pesticide Research Institute (China). Anti-OP pesticide mAb C8/D3 was produced in our laboratory.35 Mouse anti-M13 monoclonal antibodyChorse radish peroxidase (HRP) conjugate was purchased.