The Molecular Detection And Genotyping Of Fusobacterium Necrophorum

Fusobacterium necrophorum, an obligate anaerobic gram-negative bacterium, which is the major pathogen of necrobacillosis of animals as well as Lemierre's syndrome in humans. F. necrophorum is now classified into two subspecies: subsp.Necrophorum(F.n.necrophorum) and subsp.Funduliforne (F.n.Funduliforne). Subsp.Necrophorum is considered to be potentially more pathogenic than subsp.Funduliforne. We extracted bacterial genomic DNA by proteinase K method, standard NaOH-SDS lysis method and extraction kit method, determining the standard NaOH-SDS lysis method was the best extraction method of Fusobacterium necrophorum genome DNA, at last. PCR detection method and dot blot hybridization detection method were developed for detection of the genes encoding rpoB of Fusobacterium necrophorum, which can identify Fusobacterium necrophorum at the species level. we evaluate the performance of the PCR detection method by testing 28 clinical samples, 28 patients out of 19 samples testing positive, 9 negative, the detection rate was 67.8%; But, 28 patients out of 17 samples testing positive, 11 negative, the detection rate was 60.7% by dot blot hybridization detection method, which was lower than the detection rate of the PCR detection method. The detection minimum concentration of PCR detection method was 8×10-4 pg/μL; when each dot point 5μL DNA extraction solution, the detection minimum concentration of dot blot hybridization detection method was 8×10-2 pg/μL. The results indicated that PCR detection method had higher sensitivity and better accuracy than the dot blot hybridization method.Duplex PCR detection method was developed for identifying Fusobacterium necrophorum at the subspecies level. Three pairs of specific primers were designed, basing on published leukotoxin gene sequences of Fusobacterium necrophorum. L7 and L8 targeting subsp.Necrophorum and subsp. Funduliforne, respectively; L9 targeting both subspecies. Subsp.Necrophorum and subsp.Funduliforne amplify 1 076 bp and 809 bp fragments based on three primers, respectively. The detection minimum concentration of duplex PCR detection method was 0.8 pg/μL. By detecting of 17 positive clinical samples of dot blot hybridization with duplex PCR detection method, the 17 samples all was infected by subsp.Necrophorum. The duplex PCR detection method was high specificity and sensitivity, which can be used as a new tool for the differentiation of Fusobacterium necrophorum subspecies.