副溶血弧菌的磁珠捕获及检测
Bead capture and detection of Vibrio parahaemolyticus
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摘要: 建立结合免疫磁珠和PCR快速检测副溶血性弧菌的新方法。本研究选用副溶血弧菌作为抗原免疫日本大耳兔制备了多克隆抗体,用ELISA法检测抗体效价。利用辛酸硫酸铵沉淀结合逆向吸附对多抗血清进行纯化,经SDS-PAGE和斑点杂交检测验证纯化后抗体的特异性。将纯化后的抗体偶联于羧基化修饰的纳米磁珠表面,用来进行菌体的捕获。结果表明,抗体的效价达到106,纯化获得只与副溶血弧菌发生反应的高纯度抗体。制备的免疫磁珠捕获率在55%～70%之间,且稳定性较好,盐离子浓度（3.5%NaCl、4.5%NaCl）对捕获率的影响不大,在pH5～9之间,磁珠捕获差异也较小。在对纯培养物的检测实验中,本方法最低检测限为102cfu/mL;而在鱼肉糜样本中的检测限为103cfu/mL,且样品中高浓度杂菌的存在不影响检测灵敏度。免疫磁珠结合PCR用于副溶血性弧菌的检测具有很高的灵敏度和特异性,具有广阔的前景。Abstract: A novel method was developed for rapid detection of Vibrio parahaemolyticus with immunomagnetic separation followed by PCR.Whole cells of V.parahaemolyticus were used as antigen for polyclonal antibody (pAb) production in rabbit.Titers of pAb were detected by enzyme-linked immunosorbent assay (ELISA) .Antiserum was purified with ammonium sulfate precipitation and reverse adsorption, purity and specificity of the resulted pAb was tested with SDS-PAGE and dot-blot hybridization.Purified pAb was conjugated with magnetic beads for bacteria separation.Results showed that the titer of purified antibody was 106 , and the capture rates of immunomagnetic beads were between 55%～70% under different conditions.Salt concentration and pH had only slight influence on the capture rate.The limit of detection ( LOD) of the method was 102 cfu/mL in pure culture and 103 cfu/mL in fish paste samples (in presence of other kinds of bacteria species ) . In conclusion, combined immunomagnetic separation with PCR for the detection of V.parahaemolyticus has high sensitivity and specificity, which is promising for in-field detection.
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