Research And Tentative Applicaiton Of The Colloidal Gold Test Strip For Detection Of The Clostridium Perfringens A Toxin

Clostridium perfringens is a conditional pathogens widely distributed in nature, the“sudden death” which caused by Clostridium perfringens outbreaks and deaths quickly withhigh morbidity and mortality, and it had made a great damage to the animal husbandrydevelopment. AlpHa toxin is the most important virulence factor of Clostridium perfringensand each type of Clostridium perfringens can produce α toxin. At present, the diagnosis of αtoxin is mainly dependent on conventional laboratory diagnosis, but it has manyshortcomings such as the operation is complex, time-consuming and requires expensiveequipment. Therefore, a new process with high sensitivity and specificity to detect α toxinand control sudden death was very necessary. Colloidal gold chromatograpHy techniques wasconvenient, accurate, and had good specificity and sensitivity combined, it is the most widelyused diagnostic at present.AlpHa toxin expression plasmid was developed by the method of molecular biology,then the recombinant plasmid was transformed into E.coli BL21(DE3), IPTG was used forinduction to explore the optimal expression conditions; a specific band with molecular weightof45KD was appeared after the SDS-PAGE electropHoresis of expression products; a largenumber of target protein was inducible expressed under optimal conditions; after purified byNi affinity the protein was detected by Western blot and it showed that the target protein hasa good immunological activity.The expressed and purified α-toxin protein500μg was mixed with Freund adjuvant toimmune rabbits four times. The serum was obtained and the ELISA method showed that thetiter of the polyclonal antibody is1:512000by good specificity. Ammonium sulfate precipitation and dialysis bags was used to purify the antibody.According to the immune colloidal gold technique principle, we prepared detectiondipsticks using anti-α toxin polyclonal antibody as gold labeled antibody and captureantibody of the detection line to detect the Clostridium perfringens α toxin. Colloidal goldwith about40nm particle diameter was prepared by trisodium citratec reduction. The anti-αpolyclonal antibody was labeled with colloidal gold of40nm particle diameter as gold pad,and then purified the α toxin polyclonal antibody and goat anti-rabbit IgG secondary antibodyon the M135nitrocellulose membrane as the test line and control line. The specificity,sensitivity, stability and repeatability of the test strip was tested. The α toxin rapid diagnostictest strip was used to test the clinical samples and compared with the PCR results to comparethe coincidence rate.The result showed that the anti-α toxin polyclonal antibody and colloidal goldimmunochromatograpHic test strip for detection of Clostridium perfringens α toxin wasdeveloped successfully and the suitable concentration of antibody on the gold pad was1.00mg/mL; the suitable concentration of antibody on the test line was600μg/mL; theconcentration of goat anti-rabbit secondary antibody on the control online was1:20dilution;when concentration of α toxin was2.80and1.40mg/mL, detection line had a clear band;when concentration of α toxin was0.70mg/mL, the detection line strip light; whenconcentration of α toxin was0.35mg/mL, the detection line almost no band, the resultindicated that the strip has better sensitivity and specificity; tests on serum samples showedthat only Clostridium perfringens α toxin positive serum produced a positive signal on the ICstrip; the results revealed that the test strip had good specificity and reproducibility for thesame result of different batches of test strips; the detection limit of α toxin was1.40mg/mLand the correspondence rate between the colloidal gold strip and PCR was80.00%, meetingthe requirement of field application basically and had good application prospect.