魏氏梭菌α毒素快速诊断金标试纸条的研制

Development of colloidal gold immunochromatographic test strip for detection of Clostridium perfringensα toxin

【目的】研制魏氏梭菌α毒素快速诊断金标试纸条,为魏氏梭菌病的快速诊断提供新方法。【方法】采用分子生物学方法制备魏氏梭菌α毒素,将其免疫家兔制备多克隆抗体。用制备的抗魏氏梭菌α毒素多克隆抗体作为胶体金标记抗体和检测线上的捕获抗体,制备魏氏梭菌α毒素快速诊断金标试纸条,对其特异性、敏感性、稳定性和重复性进行检测。用制备的魏氏梭菌α毒素快速诊断金标试纸条对临床样品进行检测,比较其检测结果与PCR检测结果的符合率。【结果】成功制备了魏氏梭菌α毒素多克隆抗体。成功研制了α毒素快速诊断金标试纸条,其检测线上抗体的最适包被质量浓度为0.06μg/mL。研制的试纸条仅与魏氏梭菌α毒素发生反应,且不同批次的试纸条重复检测结果无差异,表明试纸条具有较高的特异性和重复性;该试纸条最低可检测1.40mg/mL的毒素量,与PCR方法测得结果符合率为80.77%,基本能满足实际应用要求。【结论】成功研制了魏氏梭菌α毒素快速诊断金标试纸条,该试纸条具有快速、特异、敏感、稳定等优点,适用于对魏氏梭菌病进行普查和检疫,具有良好的应用前景。

【Objective】 The study developed a colloidal gold immunochromatographic test strip for detection of Clostridium perfringens α toxin and provided a new method for detection of C.perfringens.【Method】 C.perfringens α toxin was developed by the method of molecular biology,and injected into rabbits to prepare anti-α polyclonal antibody.According to the immune colloidal gold technique principle,we prepared detection dipsticks using anti-α polyclonal antibody as gold labeled antibody and capture antibody of the detection line to detect the C.perfringens α toxin.【Result】 The anti-α polyclonal antibody and colloidal gold immunochromatographic test strip for detection CPT α toxin was developed successfully and the suitable concentration of antibody on test line was 1 mg/mL;Tests on serum samples showed that only C.perfringens α toxin positive serum produced a positive signal on the IC strip;The results revealed that the test strip had good specificity and reproducibility for the same result of different batches of test strips;The detection limit of α toxin was 1.40 mg/mL and the correspondence rate between the colloidal gold strip and PCR was 80.77%,meeting the requirement of field application basically.【Conclusion】 The colloidal gold immunochromatographic test strip for detection of C.perfringens α toxin was developed successfully.All these results showed that the strip was sensitive,specific,and rapid for the detection of C.perfringens,and had good application prospect.