ABSTRACT
Sulfadiazine (SD) is extensively utilized in agriculture, aquaculture, poultry, medical, and other industries. Its residues pose a threat to human health by entering the food chain and can also be released into the environment through animal feces and urine, leading to ecotoxicological pollution. Consequently, there is an urgent need to establish an efficient method for detecting SD residues in the environment. In this study, a novel two-probe fluorescence assay for determining SD in the environment, based on magnetic separation and real-time quantitative PCR-TaqMan probe technology, was successfully developed. In the experiment, SD served as the target substance, and an aptamer (Apt) with high affinity for SD was synthesized. Additionally, a non-fully complementary chain (Cdna) with favorable hybridization properties with the aptamer was designed and synthesized to create a magnetic probe of magnetic beads@Apt@Cdna. When SD was introduced, Apt specifically bound to SD with a hairpin structure and was released from the magnetic probe, allowing SD detection via the PCR-TaqMan method. Factors affecting the determination accuracy of this assay system, such as Apt concentration, SD standard solution pH, and incubation time, were optimized. Under the optimized conditions, the assay demonstrated high sensitivity for SD, with a detection limit of 2.34 × 10-5 ng/mL. Finally, the method was applied to detect SD in water samples from the Jialu River Basin in Zhengzhou City, yielding spiked recoveries of 88.82-117.05%. The results indicated that the detection system is a highly sensitive and specific method for determining SD residues in environmental water samples, showcasing its potential application in SD detection.
ABSTRACT
Tetracycline (TC) is an effective antibiotic used to treat humans and livestock, but its inappropriate use imposes toxic effects, including pollution, on environmental ecology and food. Currently, sensitive, accurate, and cost-effective methods that can detect lower concentrations of TC residues in environmental and food samples are needed. In this study, a novel indirect competitive assay-based aptamer method was developed for detecting TC residues through signal amplification by real-time fluorescence-based quantitative polymerase chain reaction. The response surface methodology was introduced to optimize the optimal concentrations (influencing factors) of the three types of single-stranded DNA in the competitive assay process. The optimal conditions for the three types of ssDNA were 112 nM for the specific aptamer of TC (Apt40), 115 nM for the signal DNA, and 83 nM for the DNA catcher. As expected, under optimal conditions, the Ct value was linearly related to the logarithm of TC concentration. The calibration curve equation was Ct = -0.34516 log[TC] + 9.9345 (R2 = 0.998) in the range of 10-3-103 ng mL-1, and the limit of detection was 7.02 × 10-5 ng mL-1. The new method was effectively applied to detect TC residues in wastewater, honey, and milk samples. It achieved an average recovery rate of 101.19% with a small variation of 5.16%. The validation was carried out using an enzyme-linked immunosorbent assay. This approach demonstrates high sensitivity and selectivity, making it well suited for detecting leftover antibiotics in food when using suitable aptamers.