Detection Of Swine Bacterial Pathogens By Reverse Dot Blot Assay

At present, the incidence of infectious diseases characterized by an increased multi-pathogen co-infection has increased the difficulty in clinical diagnosis. Rapid, specific and sensitive detection of pathogenic bacteria in weaning pigs is an important prerequisite for prevention and control of the epidemic infectious diseases. In this paper, a reverse dot blot assay has been developed for detection and identification of a variety of swine pathogens. These studies provide effective way for epidemiological investigation and so on.E. coli K88 gene sequences were downloaded from GenBank. A K88 gene specific probe was designed and added to the probe panel, a report gene that flanked with two K88 probe fragments and a pair of primer for reverse PCR was designed. Meanwhile, the probe set previously selected for the 23S rRNA based microassay was modified from 25-30 bp into 30-38 bp. The sequences of the two pairs of universal primers were remaining unchanged, except lower primers were digoxigenin-labeled. Nylon membrane was used to replace glass slide to fabricate the array, and specificity and sensitivity of the probes or RDB assay were evaluated. The encircled report gene plus reverse PCR primer pair were added to the original PCR system to prepare target amplicons for RDB assay.The modified probes and the RDB were proved specific when assessed with 13 reference target strains. The detection limit of the assay was determined as approximately 100 fg crude DNA, equivalent to the previous gene chip. Testing with 5 non-target reference strains and 34 field isolates showed that the correct identification was 35/39 (89.7 %), also close with the results of previous gene chip. The RDB based on triplex PCR allow simultaneous detection of common 23S rRNA and E. coli K88 gene. The study showed that the RDB assay is a simple,rapid and cost effective alternative for slide based microarray on detection and identification of swine bacterial pathogens in laboratories with limited equipments and funds. It could also improve diagnostic ability by integrating virulent or pathogenic gene probes and reverse PCR method into the assay system as demonstrated in this study.These studies may provide a useful platform for clinical diagnosis...