An aptamer-based effective method for highly sensitive detection of chloramphenicol residues in animal-sourced food using real-time fluorescent quantitative PCR.
Chloramphenicol (CAP) residues can not only harm human health through entering food chain, but also cause the spreading of drug-resistant bacteria, thereby leading to secondary environmental pollution. Therefore, it is in urgent need of establishing an efficient technology to detect CAP residues in animal-sourced food. In this study, a novel sensitive approach for detection of CAP was designed based on a CAP specific aptamer and real-time fluorescent quantitative PCR (qRT-PCR). The CAP specific aptamer was firstly hybridized with a biotin modified complementary probe, and then was immobilized on streptavidin conjugated magnetic beads through biotin. When CAP was added, the aptamer would specifically bind with CAP by forming a hairpin structure and be released from the magnetic beads for CAP detection by qRT-PCR. Factors (i.e., probe strand length, aptamer concentration, NaCl concentration and incubation time) that would influence the determination accuracy of this aptamer-based detection system were optimized. Under the optimized conditions, the present detection system exhibited a high sensitivity toward CAP with a limit of detection of 0.1ng/mL (linear range from 0.1 to 20ng/mL). Moreover, this detection system also showed high selectivity against thiamphenicol (TAP) and florfenicol (FF), which are CAP's structure analogs. Eventually, this detection system was applied for detecting CAP in real spiked milk. The recovery rate of CAP from spiked milk samples ranged from 94.0-102.0%. These results indicated this developed detection system a promising high sensitive and specific method of CAP residues detection in animal-sourced food.
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
Ye
Y
Duan
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
Lihui
L
Wang
Beijing Entry-Exit Inspection and Quarantine Bureau, Beijing 100026, China.
Zhiqiang
Z
Gao
Beijing Senkang Biotechnology Development Co., Ltd, Beijing 101400, China.
Huishan
H
Wang
Beijing Entry-Exit Inspection and Quarantine Bureau, Beijing 100026, China. Electronic address: zhanghx@bjciq.gov.cn.
Hexiao
H
Zhang
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China. Electronic address: lihao@mail.buct.edu.cn.
Hao
H
Li
D000821:Animal Feed / Q000032:analysis*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis / Q000737:chemistry; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D002417:Cattle; D002701:Chloramphenicol / Q000032:analysis* / Q000737:chemistry; D004350:Drug Residues / Q000032:analysis* / Q000737:chemistry; D008892:Milk / Q000737:chemistry*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
D000900:Anti-Bacterial Agents; D052157:Aptamers, Nucleotide; D002701:Chloramphenicol
Aptamer-based detection system; Chloramphenicol; Milk analysis; Real-time fluorescent quantitative PCR; Specific recognition