Establishment And Application Of Rapid Immunochromatographic Methods For The Detection Of Fluoroquinolone Residues In Livestock And Poultry Meat

Fluoroquinolones(FQs)belong to a new generation of chemically synthesized quinolones derivatives,which are widely used as antibacterial drugs to prevent and treat infectious diseases in animals.However,due to the unreasonable use of FQs by humans,problems such as veterinary drug residues,increased bacterial resistance,and environmental pollution have been caused,posing a huge threat to human health.In recent years,China has issued a series of Maximum Residue Limits(MRLs)standards for FQs in food to regulate the use of FQs.In 2016,China decided to stop the use of lomefloxacin(LOM),Pefloxacin(PEF),Ofloxacin(OFL)and Norfloxacin(NOR)in food animals.In 2022,the Maximum Residue Limits of 41 veterinary drugs in food safety national standard foods(GB 31650.1-2022)was issued to further improve the provisions on the prohibition of FQs.However,according to statistical data released by relevant departments in recent years,the detection of prohibited FQs such as OFL in the main meat products consumed in China(pork,chicken,and beef)still occurs from time to time,and the proportion of enrofloxacin(ENR)residues exceeding the limit in the detected FQs is relatively large.Therefore,it is urgent to establish a fast and sensitive analytical method to detect the residues of four prohibited FQs and ENR in pork,chicken,and beef.Compared with instrumental analysis methods,lateral flow immunoassay(LFIA)has the advantages of speed,simplicity,and low cost,and has been widely used in fields such as food safety,medical diagnosis,and environmental testing.The traditional LFIA uses colloidal gold(CG)as the signal output carrier,which is mature and simple in technology,and the colorimetric signal is easy to directly interpret with the naked eyes,making it suitable for on-site rapid and qualitative detection.The new fluorescent LFIA uses different fluorescent materials as signal markers,which have advantages such as high sensitivity and resistance to matrix interference,and can be used for quantitative analysis of target substances.The strategy of using aggregation induced emission fluorescence microsphere(AIEFM)as a marker solves the problem of fluorescence materials being prone to quenching under high loading,further improving the performance of LFIA.This study established two AIEFM-LFIAs based on two monoclonal antibodies(m Ab)for quantitative detection of prohibited FQs and ENR in pork,chicken,and beef,respectively.Two CG-LFIAs were also established using the aforementioned m Ab for qualitative detection of prohibited FQs and ENR in three types of meat products.The first chapter provides a systematic overview of the detection target FQs,including their physical and chemical properties,mechanism of action,current application status,toxicity hazards,domestic and foreign limit standards,and commonly used FQs residue detection technologies.Next,a brief introduction will be given to LFIA,including its principle,markers,and sampling method.In Chapter 2,AIEFM-LOM-LFIA was established based on the laboratory prepared broad-spectrum specific anti-LOM m Ab for quantitative detection of prohibited FQs in meat products;AIE-ENR-LFIA was established based on purchased commercial anti-ENR m Ab for quantitative detection of ENR in meat products.Relevant parameters were optimized in the experiment,including p H,antibody labeling amount,the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxy succinimide,the complete antigen concentration of the detection line,the amount of immune probe spray,sample adding diluent and sample extraction agent.The results showed that under optimal conditions,the limit of detection(LOD)of LOM in pork,chicken,and beef detected by AIEFM-LOM-LFIA were 0.187,0.068,and 0.057 μg/kg,respectively.The cross reaction rates of AIEFM-LOM-LFIA for PEF,OFL,and NOR were 54.4%,31.5%,and 49.2%,respectively The recovery rates of the four prohibited FQs in the three meat foods were between 73.1%-125.90%,and the coefficient of variation(CV)was between 0.5%-23.66%;Under optimal conditions,the LOD of ENR detected by AIEFM-ENR-LFIA in pork,chicken,and beef were 9.555,7.217 and 4.809μg/kg,respectively.The spiked recovery rates of ENR in the three types of meat products at g/kg range from 70.11% to 120.62%,with CV ranging from 1.63% to19.36%,and the specificity of AIEFM-ENR-LFIA is good.The combination of AIEFM-LOM-LFIA and AIEFM-ENR-LFIA can achieve sensitive and quantitative detection of LOM,PEF,OFL,NOR and ENR in three types of meat products.In Chapter 3,CG-LOM-LFIA and CG-ENR-LFIA were established based on the above two antibodies,respectively,for the qualitative detection of prohibited FQs and ENR in meat products.Relevant parameters were optimized in the experiment,including p H,antibody labeling amount,complete antigen concentration in detection line,immune probe spray amount,and sample extraction agent.Under optimal conditions,CG-LOM-LFIA was used to qualitatively detect LOM,PEF,OFL,and NOR in three types of meat products,with naked eye detection thresholds of 0.5,1,2 and 3,respectively μg/kg.Qualitative detection of ENR in three types of meat products using CG-ENR-LFIA under optimal conditions,with a naked eyes detection threshold of 50μg/kg.The combination of CG-LOM-LFIA and CG-ENR-LFIA can achieve rapid qualitative detection of LOM,PEF,OFL,NOR and ENR in three types of meat products.Chapter 4 provides a systematic summary of the four quantitative and qualitative LFIAs established in this article for detecting FQs,and looks forward to new ideas for improving LFIA performance and preprocessing techniques in the future.In summary,this study established a quantitative and qualitative LFIA for detecting four prohibited FQs and ENRs in pork,chicken,and beef,providing strong technical support for grassroots regulatory authorities to monitor the four prohibited FQs and ENRs in pork,chicken,and beef.