Development Of An Up-converting Phosphor Technology Based Lateral Flow Assay For Quantitative Detection Of Burkholderia Pseudomallei And Fra Ncisella Tularensis

ObjectiveTo develop a highly sensitive and robust, culture-independent, up-convertingphosphor technology-based lateral-flow (UPT-LF) assay that allows rapidly directquantification of Burkholderia pseudomallei and Francisella tularensis from complexsamples on site.MethodsUPT-LF strips for B. pseudomallei and F. tularensis were developed based on thedouble-antibody sandwich mode by using specific monoclonal antibodies against B.pseudomallei and F. tularensis, respectively. Then sensitivity, linearity and specificity ofthe strips tests were assessed by using bacterial suspension with serial standardconcentrations. For on-site detection, tolerance of the test was evaluated by using seriesconcentrations of pure chemical reagents (HCl, NaOH, NaCl+KCl, PEG20000, glycerin,etc) and several kinds of simulated samples (flours, milk powder, greasy powder, soil,organs etc.). Furthermore, operation-error tolerance of volume variation including samplevolume variation,treating buffer volume variation and total detecting sample volumevariation during onsite detection were also considered and evaluated in this study.Results1. After being optimized, the monoclonal antibody1B6(3mg/ml) against B.pseudomallei and goat anti mouse IgG (3mg/ml) were dispensed on the nitrocellulosemembrane as the test line (T) and control line (C) of the UPT-LF strip for B. pseudomallei,while monoclonal antibody mixtures (2E5,3D6and8G1) against B. pseudomallei werelabeled with up-converting phosphor (UCP) particles. For UPT-LF strip for F. tularensis, monoclonal antibodies4H10and1C5(2mg/ml) against F. tularensis and goat anti mouseIgG (2mg/ml) were employed as the T line and C line, while monoclonal antibody12H1against F. tularensis were labeled with UCP particles.2. The detection limit of the strip test for B. pseudomallei was104cfu/ml. The linearfitting coefficient (r) was0.996with bacterial concentrations ranging from104cfu/ml to107cfu/ml. And the assay showed no cross reactions with30other bacteria including bacterialbiowar agents, foodborne pathogens and other related bacteria. For UPT-LF strip for F.tularensis, the detection limit was104cfu/ml. The linear fitting coefficient (r) was0.995with bacterial concentrations ranging from104cfu/ml to108cfu/ml. The assay showed nocross reactions with18other bacteria.3. Both of the UPT-LF tests can keep robust with series concentrations of purechemical reagents (HCl, NaOH, NaCl+KCl, PEG20000, glycerin, etc). The simulatedpositive samples (flours, milk powder, greasy powder, soil, fruit juice, etc.) also had noobvious effect on the performance (detection limit, quantification capability and specificity)of the tests. Given the operation error including target sample volume、 treating buffervolume and total detecting sample volume, performance of the UPT-LF tests for B.pseudomalleiand F. tularensis changed little.ConclusionThe research developed two kinds of UPT-LF assay for quantitative detection of B.pseudomalleiand and F. tularensisis respectively with high sensitivitye， specificity andtolerance. The excellent performance promised the bright future of UPT-LF assay fordisease prevension and control and anti-bioterrorist response.