乳胶凝集试验快速检测副猪嗜血杆菌

A Latex Agglutination Test for Detection of Haemophilus Parasuis

利用原核表达并经过纯化的副猪嗜血杆菌TbpBN端蛋白及人工合成的特异性多肽制备单克隆抗体,经碳化二亚胺(EDC)将纯化后的单克隆抗体IgG与羧化乳胶共价偶联,并对偶联乳胶的IgG含量及偶联时间等条件进行合理选择,建立了快速检测副猪嗜血杆菌的乳胶凝集试验方法。利用LAT方法检测148株副猪嗜血杆菌疑似菌株,参考16S rRNA-PCR方法的结果,符合率为83.1%,且致敏乳胶不与临床常见菌株发生凝集反应。结果表明,此检测方法具有操作简便、快速、特异性高的特点,便于在基层推广使用,为副猪嗜血杆菌的诊断和有效防治提供了参考和依据。

A Latex Agglutination Test （LAT） was developed for quick detection of Haemophilus parasuis . Recombinant TbpB-GST protein of Haemophilus parasuis was obtained and purified, as welt as a unique 91 bp polypeptide within TbpB N-terminal domain from position -226 to -318 was synthesized. Afterwards, two groups of six to eight-week old female Balb/c mice each was immunized with the recombinant fusion protein and the peptide conjugated with KLH （keyhole limpet hemocyanin）. Two individual mono- clonal antibodies IgGs were produced and covalently linked to carboxylated polyethylene latex beads by EDC. The amount of IgGs and coupling time were optimized for LAT test. 148 field strains isolated from lungs, spleens, arcula cordis, articular cavities and brains were detected by LAT and 1G S rRNA-based PCR. The result demonstrated that the coincident rate was 83.1% between PCR and LAT test. In addi- tion, sensitized latex particles （SLPs） were not agglutinated by heterologous strains of Pasteurella rnultocida , Actinobacillus pleuropneumoniae , Escherichia coli , Staphylococcus aureus or Streptococcus . In summery, Latex agglutination test is a very simple, rapid and effective method and it could be used in field investigations.