Studies On Multiple Detection Technology Of Vibrio Choleraeusing Up-converting Phosphor Technology-based Immunochromatographic Assay

1.Background and ObjectivesCholera,an acute diarrheal disease,is caused by Vibrio cholera.Its acute onset,highlycontagious and high mortalitymakes this disease ranked as class “A” infectious disease by the " Infectious Disease Prevention Law of People’s Republic of China ".Now the cholera epidemic is still reported,putting a serious threat to humans’ health.Therefore,fast,simple and accurate detection of cholera is critical for prevention and control of this disease.Vibrio cholerae has more than 200 serogroups,of which groupsO1 and O139 are the main one to causing human epidemics,and other groupsarerarely associated with epidemics.Hence,accurate detection of groupsO1 and O139 has great practical significance.The existing methods of cholera detection are more or less withsome problems,such as some need to be performed by experienced operators,and some be reliedon sophisticated equipment,and some easily be lead to misjudgment,some can only give qualitative results,some take tedious operation with a long time,some are with lower sensitivity,and some cost higher.These drawbacks showed an urgent requirement for development of rapid test to detect Vibrio cholerae.2.MethodsImmunochromatography,as a classic fast test technology,is easy to operate by adding sample directly to the sample pad,and putting it aside for 10 to 15 minutes,then,wecan readthe results withour naked eyes if the chromatographic assay is based on gold as a label.For the classic gold-immune chromatographictechnology,it usuallydependson the operator’s personal experience,can only give qualitative result,and easily impacted by assay parameters,including salt,pH,and many others,restricting its wide application for detecting targets in “dirty’ or “contaminated”samples.Combining upconversion phosphor materials with immunochromatography can overcome theseproblems to a large extent.The results show that the advantages of the classical immunochromatographic technique,such as the simple operation withhandle-held equipment,while solving the disadvantages of time-consuming and non-quantitative detection for the classical immunochromatographic methods.In this paper,three test strips were prepared by double-antibody sandwich method based on UPT-ICG for simultaneous detection of the Ogawaserogroup,the Vibrio cholerae O1 group and/or the Vibrio cholerae O139 group,with three detecting bands without any interferencebyeach other.By optimizing the composition of the strips,the combination of the amount of the pad antibody and the antibody-labelling conditions were determined,and the amount of the membrane antibody was alsotitrated.Finally,the three kinds of single target stripsand one kind of three-target stripswere prepared for evaluating their specificity,sensitivity,linearity and precision.The strips were also evaluated by using different bacterial isolates.3.ResultsWe prepared the specific monoclonal antibodies specific for different groups of Vibrio cholerae,includingmonoclonal antibody 9B12 for Ogawa serogroup,9F11 for Inaba serogroup,and antibody mAb1 and mAb2 for O139.The above-mentioned three strips for detecting respectively the Ogawa serogroup,the O1 group of V.choleraeand O1 and/or O139 serogroup were prepared.On the basis of three kinds of single-target strips,we developed a three-target stripthat could simultaneously detects different serogroups ofV.cholerae.The sensitivities for the stripsto detect Ogawa serogroup or O1 group of V.cholerae reached 102cfu/ml,and for other strips 103cfu/ml.There was no non-specific reaction with other food-borne pathogens,including Salmonella,Vibrio parahaemolyticus,pathogenic E.coli,and Listeria etc.These strips were evaluated using different strains isolated from China in collaboration with China CDC.The results demonstrated that the strips developed by us is specific,sensitive,accurate,and easy-to-operate.The results were quantitative,showing promising in future applications.4.ConclusionGiven the fact that the existing Vibrio cholera detection methods cannot fully meet the requirements of clinical rapid test and there are a variety of defects in different assays.In order to develop a feasible rapid,quantitative assay to detection different serogroups of Vibrio cholerae on site,we applied up-conversion phosphor technology(UPT)to immunochromatography(ICG)to successfully establish aUPT-ICG rapid detecting technology.We used this technology for detecting V.cholerae,the developed strips showed strong specificity,high sensitivity and accuracy,robustness with contaminated samples.This method gives final quantitative results within 15 minutes and the results are showed by digital reports.The biosensor for detecting the results is easy to carry.This technology could also be used in other situations,such as clinical emergency,disaster rescue,detection of drug-abuse and anti-bioterrorism detection.