Development Of Immuno-chromatogaphic Assay Applied In Point-of-care Testing For The Diagnosis Of Foot-and-mouth Disease Virus

"Point-of-care testing"(POCT) refers to the use of the procedures oflaboratory medicine in the immediate vicinity of the patient The key advantages ofPOCT are that it investeigations can be performed rapidly and simply, the results areimmediately available at the patient's bedside. reagent is stable and easy to preserveand carry.POCT is increasingly used in the clinical laboratories in recent years. footand mouth disease (FMD) is one of the most important known contagious animalvirual disease, Outbreaks can severely disrupt livestock production and result inembargoes by trade partners, and caused direct and indirect economic losses to benot uncommon Thereforce the accurate and prompt diagnosis and routinesurveillance become very important for the disease control and prevention of virusincursion. routine diagnostic methods include virus isolation from cell culture,ELISA, and reverse-transcriptase polymerase chain reaction (RT-PCR) whichrequirewell-trained laboratory personnel, special laboratory and expenseiv instruments inpractice.Thereby offering the possibility of implementing control procedures morerapidly and conveniently has become an urgent problem to be solved, and it will bepromsing for future diagnosis.Combination of nanotechnology and someconventional technologies such as lateral flow test makes immuno-chromatogaphicassay demonstrate enormous superiority in POCT. The aim of this study wasdeveloping and evaluating an immuno-chromatogaphic assay using a colloidal goldand latex particles as indicators for typing of FMDV antigen,quantification ofFMDV antibody and nucleic acids testing as well as The development prospects ofrapid diagnosis dipsticks in POCT were discussed.1. Development of a rapid gold immunochromatographic strip test for thediagnosis of Foot-and-mouth Disease Virus O, A and Asia1TypeMost research focused on facilitating type-specific pen-side diagnosis todifferentiate FMDV and other vesicular viral pathogens by using FMDV serotype-specitic monoclonal antibodies, however,little report is on the developmentof serotyping diagnostic kits for FMDV. In this part, andtibodies obtained fromrabbits and guinea pigs immunized with cell-culture-adapted virus strains(O/CHA/99, A/GS/LX/66, Asia1/CHN/05) and suckling-mouse adapted virus strains(O/AV99(L), A/AV88(L), Asia1/YNBS/58) were used as capture antibodiesaccording to control of Characterization of the colloidal gold probe, optimizedantibody concentration and pH of conjugations colloidal gold,selected adsorptionprotein fixed and dissolved to nitrocellulose,it was development of a simple andrapid LFI for the detection of FMDV O, A and Asia1serotypes in samples fromanimal vesicular and epithelial fluids. The seriate results indicated that the sensitivityof the test kit can reach to7.8×104LD50.(128-diluted mouse tissue-derived virus)And it had the same results for positive and negative samples tested in triplicate. Nocross reaction was found with swine vesicular disease (SVD),VS,VES antigen. bycross tests. In the clinical assay, the corresponding rate of goldimmunochromatographic strip test kit for FMDV O,A,Asia1type and negativesample was92.45%,91.66%,92.75%and100%respectively. The coincidence rate ofdetected FMDV serotype was94.58%. It is the fist report on the development of anLFI for typing FMDV serotypes O, A and Asia1which has been awarded the stateinvention patent.2.Development of a rapid colour latex particles immunochromatographic striptest for the typing of Foot-and-Mouth Disease virusTo match a repuirement of multi-parameter detection for the typing of FMDVwith the limited samples.It was developed of colour latex particlesImmunochromatography assay for determination of foot-and-mouth diseasevirus(FMDV) O, Asia1type from the field samples. Monodispersed polystyreneMicrospheres with two distinct dyes and carboxyl group in the size rang of380nmwere prepared with seed polymerization and swelling. The purified anti-FMDVstrain Asia1/XJ/AKT/03, O/GD/NX/92rabbit antibodies were separately coupledwith blue and red latex particles. The purified anti-Asia1/YN/BSh and AV(99)L FMDV rabbit antibody were wrapped on to nitrocellulose membrane as two test line.The FMDV serotype diagnostic kit was then performed and conditions weredetermined. The results indicated that sensitivity of the test kit reached3.9×104LD50,and no cross reaction was found with SVD,VS,VES antigen by cross tests. In theclinical assay, a total74field samples were detected with the test strips. The result ofpositive samples for FMDV O, Asia1type and negative samples were95.24%,96.30%,100%respectively.3. Development of a rapid gold immunochromatographic strip test detecting theantibodies against FMD type OAlthough it has been repoted at2004that rapid chromatographic srip detedtedthe antibodies of serotype O.the assay could only detect the antibodies from swinesera and was not suitable for the other animals such as cattle and sheep.In the study,using inactivated146S antigen of two strains of serotype O FMDV which come fromswine and cattle cell bath respectively. One of antigen was labeled with colloidalgold, dispensed onto fiber glass. Another one was separately dispensed onto an NCmember to form a test line The diagnostic kit was employed to detect the antibodiesagainst FMD type O viruses from swine, cattle and sheep. According to the antibodytiters of liquid-phase blocking sandwich ELISA(LB-ELISA) and chromogenicsituation of gold Immunochromatography assay(GICA), the colourimetric card ofGICA was drawn. Which improved antibody titer evaluation from the qualitation tohalf quantitative. The sensitivity of the GICA for all samples was confirmed to be95.3%, and the The result of specificity for FMDV O, Asia1type and negativeserum was91.4%,93.3%and100%respectively. Using a1:8dilution of the sample,if the GICA result was positive, it meant the inoculated animal serum had serotype OFMDV immune protective antibodies. Howevre,if the result was negative, and theanti-FMDV serum was belonged to non-protection rang. This technology has alreadyapplied for patent invention, and is under the review procedure.4.Development of oligonucleotide lateral-flow immunoassay for detection nucleicacid of PCR amplification In POCT asssys, nucleic acid plays one of irreplaceable roles as it canreflectintuitively disease status and the severity of the virus infection.It will be adevelop a new application of immuno-chromatograpic strip. Nucleic acid lateral flowimmunoassay(NALFIA) that is a simple, sensitive and specific detection system. foramplified FMDV3D gene by PCR was described in the study. An ultrasensitivenucleic acid biosensor(NAB) based on streptavidin-labeled gold nano-particles duallabels and lateral flow strip biosensor(LFSB) was used in this system. The NALFIAwas evaluated using gel-electrophoresis analysis. The detection lower limit ofNALFIA was0.4-1pg/mL DNA in the template and8.28μg/mL product of PCR.Ithad an excellent consistency between gel-electrophoresis and NALFIA. Inconclusion, it was a good alternative to detect PCR product of FMDV3D gene.