| Escherichia coli K88(ETEC K88),as the most common enterotoxigenic Escherichia coli strain,is the main pathogenic microorganism of piglet diarrhea,and also one of the causes of human diarrhea(adults and children).Especially in some developing countries,the infection probability of ETEC K88 is more common in infants with diarrhea symptoms.In the breeding industry,in order to improve economic benefits and prevent animal diseases,antibiotics are often used as a quick and effective method in the treatment of piglet diarrhea.Chloramphenicol(CAP)is a kind of high-efficiency broad-spectrum antibiotics with low price and good bactericidal effect,which has been widely used in the control of pathogenic bacteria in the breeding industry.CAP residue is potentially harmful to human body and has been listed as a prohibited drug in most countries,but it is still illegally used in animal breeding.A large number of antibiotics,such as chloramphenicol,are used illegally,resulting in serious bacterial resistance and antibiotic residues in animal derived food.Therefore,the establishment of a rapid,convenient and accurate ETEC K88 detection method is of great significance to the scientific and effective prevention and control of piglet diarrhea caused by it,and to reduce the use of antibiotics as well.In addition,it is still necessary to establish a highly sensitive and selective detection method for chloramphenicol in order to improve the safety of animal derived food.Aptamer is a single stranded oligonucleotide fragment screened by SELEX technology.Although it has no genetic information,it can form a unique three-dimensional structure and bind to the target specifically.Because of its advantages of good specificity,easy modification and other advantages,it is widely used in pathogen detection,food safety and other fields.Graphene oxide(GO),which has a wide wavelength absorption spectrum and high quenching efficiency,is often used as a fluorescence quenching agent.GO can adsorb ss DNA,but does not adsorb ds DNA and aptamer target complex,so it can be used as a carrier.Therefore,GO is an ideal material for fluorescent sensors.As a kind of signal amplification system,biotin streptavidin has been applied in many fields because of its high affinity and good stability.Based on the aptamer and biotin streptavidin reaction system,a microplate fluorescence probe detection system was constructed to detect ETEC K88 in water.At the same time,with chloramphenicol as the target,fluorescent dye as the fluorescence donor,and GO as the fluorescence resonance energy transfer carrier,the fluorescence aptamer probe detection platform and fluorescence sensor system were designed and applied to the detection of chloramphenicol in pig urine.The specific research contents are as follows(1)Establishment of microplate fluorescence probe detection system for ETEC K88.The 5’end of ETEC K88 aptamer was labeled with FAM as fluorescence capture probe FAM-Apt-K88,and the 5’end of its partially complementary sequence was labeled with biotin as fixed probe Bio-Apta-P.In the absence of target,FAM-Apt-K88 and Bio-Apta-P can bind to streptavidin labeled microplate after forming double chains,and there is no fluorescence signal in the system.When a target exists,FAM-Apt-K88 can be separated from Bio-Apta-P,and the fluorescence signal can be detected again.It was displayed that fluorescence intensity and logarithm of ETEC K88 have a good linear relationship(R~2=0.9801),and the detection limit is 1.36×10~3 CFU/m L when the concentration of ETEC K88 was in the range of 1.36×10~3～1.36×10~8CFU/m L.Therefore,It should successfully applied to the detection of ETEC K88 in water.(2)Establishment of a fluorescence aptamer probe detection platform for chloramphenicol.The probe Pro-Apt-L was constructed by adding some random bases and T bases to the 3’end of chloramphenicol aptamer,which could be hybridized with the fluorescent aptamer probe FAM-Pro-Apt-L containing complementary bases.The 5’end of FAM-Pro-Apt-L is labeled with FAM,with a poly T structure and four random base sequences as the interval,and two t bases are added at the 3’end as the interval.Poly T structure and random base sequence can make Pro-Apt-L and FAM-Pro-Apt-L better retain their single chain extension and enhance the quenching efficiency of GO.When chloramphenicol was added,Pro-Apt-L combined quickly with it to form a double chain complex,detached from the surface of GO,and the fluorescence signal in the FAM-Pro-Apt-L was restored and could be re-detected.Under the optimized conditions,the linear range of detection was 10–60μg/L,and the detection limit was 0.5μg/L for chloramphenicol.Furthormore,their recoveries and reproducibility were 83.9%-93.6%and 5.5%-7.2%,respectively.(3)Construction of chloramphenicol fluorescence sensor detection system.The capture probe(capture probe)with recognition ability,and the fluorescent signal probe I(signal probe I)and signal probe II(signal probe)binding to it complementary were designed respectively.The capture probe consists of a chloramphenicol aptamer and a random base.The signal probe is composed of random complementary bases and labeled with hydroxyl fluorescent group at one end.After complementary connection between capture probe and signal probe I,signal probe II,the fluorescence signal is significantly amplified.In the absence of CAP,both the signal probe and the capture probe are in a flexible single chain state,which are adsorbed on the surface by GO throughπ-πinteraction,resulting in the fluorescence signal of FAM being quenched by FRET mechanism.When CAP was added,the capture probe and CAP formed a complex and separated from the GO surface,and the fluorescence signal recovered.Under the optimized experimental conditions,the fluorescence sensor system has a good linear detection of chloramphenicol in the range of 20-200μg/L(R~2=0.9123),and the detection limit was 0.3μg/L.Additionally,the recoveries and reproducibility in pig urine samples were97.73%-108.56%and 4.66%-8.90%,respectively. |
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