Research On Immunochromatographic Assay For Detecting Clenbuterol And Escherichia Coli O157:H7

Immunochromatographic assay(ICA)is widely applied in the field of on-site testing because of its simple,rapid,high sensitivity,strong specificity and low cost.However,traditional gold nanoparticle-based immunochromatographic assay(GNPICA)is always limited by its qualitative mode.What's more,a visually observation the presence of the test line is always required to determine the test results.Thus,the test results are susceptible to subjective factors.The ICA based on reader mode has become the focus of research in recent years because of its simple operation,high sensitivity,and effortlessly storage or uploading data.The aim of this study is to established a rapid semi-quantitative detection system of GNP-ICA for semi-quantitative detection of Clenbuterol(Clen)residues in pig urine and beef.The constructed system basically avoid the influence of the environment and the subjective factors on the test results.This study further systematically compared the performance of GNP-ICA,quantum dot-based ICA(QD-ICA),fluorescent nanoparticle-based ICA(FNP-ICA),and Eufluorescent nanoparticle-based time-resolved ICA(EuNP-ICA)in quantitative detection of Escherichia coli O157:H7(E.coli O157:H7).In order to verify the practical application value of the pig urine sample and the beef sample by using the semi-quantitative GNP-ICA established in this study.256 pig urine samples from different regions,which were confirmed to be negative by ELISA,were spiked to study the negative / positive distribution and the false negative / positive rate.At the same time,80 spiked beef samples were studied by blind sample test.The false negative / positive rate were analyzed.As a result,the false positive / negative rate near the sensitivity point was relatively high,while the negative samples were not detected false positive,and the strong positive samples were not detected false negative.This study offered a reference for the GNP-ICA based on the reader model for practical detection.ICA based on novel nanoparticles with photoluminescence properties have become the focus of research in recent years.Novel ICAs based on photoluminescent nanoparticles have been developed to improve the sensitivity of target.In this study,GNP,QD,FNP,and EuNP were used as ICA labels.The abilities of the four ICAs to quantitatively detect E.coli O157:H7 in milk were first systematically compared.Various parameters influencing the four ICAs were optimized.The sensitivities of GNP-ICA,QD-ICA,FNP-ICA,and EuNP-ICA to E.coli O157:H7 were 2.5×104,5×103,1.0×103,and 5.0×102 colony-forming units(CFU/m L),respectively.EuNP-ICA exhibited the highest sensitivity.The amounts of monoclonal antibody(mAb)per GNPICA,QD-ICA,FNP-ICA,and EuNP-ICA test strip were 0.16,0.37,0.04,and 0.10 ?g,respectively.The corresponding CV were 7.4%–15.8%,10.4%–18.6%,2.7%–7.8%,and 6.9%–10.5%.FNP-ICA required the least amount of mAb per test strip and had the smallest CV.The linear ranges of GNP-ICA,QD-ICA,FNP-ICA,and EuNP-ICA were 1.0×104–1.0×106,2.5×103–1.0×106,2.5×102–2.5×105,and 2.5×102–2.5×105 CFU/mL,respectively.FNP-ICA and EuNP-ICA had wider linear ranges than GNP-ICA and QDICA.Additionally,FNP-ICA and EuNP-ICA showed better tolerance than GNP-ICA and QD-ICA during the detection of the milk sample.FNP-ICA and EuNP-ICA showed remarkable potential for the detection of pathogens in milk.