用反向斑点杂交方法检测猪常见致病菌

目的:建立检测猪常见致病菌的反向斑点杂交方法。方法:将23S rRNA基因芯片用的针对12种细菌的25～30 mer探针加长到30～38 mer,2对通用引物序列不变。用地高辛标记下游引物,以尼龙膜为载体制备膜芯片,检验探针/膜杂交的特异性和敏感性;另外设计1条大肠杆菌K88基因探针、一段带K88探针的报告基因和1对报告基因的反向PCR引物,在PCR体系中增加封口的K88报告基因和反向引物对,被检样品扩增后进行膜杂交。结果:修改的13条探针与参考目标菌株在膜上成特异性杂交,对52个参考菌株和野外分离株的检测准确率为92%;膜杂交的敏感性与玻片芯片接近,最小检出量为100 fg DNA;在尼龙膜上增加K88探针,与3重PCR产物杂交,可以检测到大肠杆菌K88毒力基因。结论:建立的反向斑点杂交方法简便快速,检测成本低,可用于仪器设备不足的实验室,同时可以加入检测如大肠杆菌K88等致病基因,提高基于保守基因的芯片的诊断能力。

Detection of Swine Bacterial Pathogens by Reverse Dot Blot Assay

Objective：To develop a reverse dot blot（RDB） assay for detection of swine pathogenic bacteria.Methods：The probe set previously selected for the 23S rDNA based microassay was modified from 25～30 mer in-to 30～38 mer.The sequences of the two pairs of universal primers were remaining unchanged,except lower primers were digoxigenin-labeled.Nylon membrane was used to fabricate the array,and specificity and sensitivity of the probes or RDB assay were evaluated.Meanwhile,a K88 gene specific probe was designed and added to the probe panel,a report gene which flanked with two K88 probe fragments and a pair of primer for reverse PCR was designed to amplify the report gene.The encircled report gene plus reverse PCR primer pair were added to the o-riginal PCR system to prepare target amplicons for RDB assay.Results：The modified probes and the RDB were proved specific when assessed with 13 target and 5 non-target reference strains and 34 field isolates.The correct identification was 48 / 52（92%） in total.The detection limit of the assay was determined as approximately 100 fg crude DNA,equivalent to the previous gene chip.The RDB based on 3-PCR allowed simultaneous detection of common 23S rDNA and K88 gene of E.coli.Conclusion：The results showed that the assay is a simple,rapid and cost effective alternative for slide based microassay on detection and identification of swine bacterial pathogens in laboratories without probe spotter and scanner.It could also improve diagnostic ability by integrating virulent or toxic gene probes and reverse PCR method into the assay system as demonstrated in this study.