Construction Of Real-time Taqman PCR Rapid Diagnosis Method Of Yersinia Enterocolitica And Analysis On Polymorphism Of Urease Gene And Urease Activity

Yersinia enterocolitica is gram negative enteropathogen transmitted by consumption of contaminated water or food. Yersinia enterocolitica could cause some gastrointestinal syndromes as well as sequelaes such as reactive arthritis, erythema nodosum and Yersinia hepatitis which cause worldwide concern. Yersinia enterocolitica has the ability to multiply at low temperatures and in vacuum packages. Not only many Yersinia enterocolitica infections but also some Yersinia enterocolitica outbreaks have been reported worldwide. In order to estabilish a more sensitive and faster method to identify yersinia entercolitica and further probe factors related to yersinia entercolitica virulence, we designed TaqMan probes and primers based on the research for ail and foxA genes. It is the first time we developed a Real-time Taqman PCR method which is based on ail and foxA genes for simultaneous identifying pathogenic Yersinia enterocolitica as well as none pathogenic Yersinia enterocolitica. In addition, we cite a internal amplification control to exclude false-negative result.It is found that the method we estabilished in this study has 100% specificity and high sensitivity. The internal amplification control could detect false-negative result efficiently and do not affect amplification result of the sample. Furthermore, when it is used to identify on separate specimens, real-time PCR method is not only consistent with traditional culture method and conventional PCR method but also more sensitive and convenient. Real-time PCR method improves the purpose of traditional culture. So it is possible to substitute conventional PCR method by Real-time Taqman PCR method we estabilished in this study for preliminary screening before traditional culture method.In addition, we chose 43 Yersinia enterocolitica strains, which had different pathogenicity. After PCR and sequencing, we conducted sequence alignment with 4 NCBI reference sequences and constructed cluster tree. Then we chose 8 Yersinia enterocolitica strains which had different pathogenicity to perform urease activity experiment. So that, we could investigate the polymorphism of Yersinia enterocolitica urease gene and the correlation of urease activation between different pathogenicity strains. The results demonstrated that seven ORF of urease gene complex were highly conserved among serotype 0:3 strain and serotype 0:9 strain respectively. Urease gene of NX1997-IE419 may evolve from serotype 0:3 strain. Sequences of high-pathogenic strain and non-pathogenic strain were not conserved. Urease activity experiment suggested that pathogenicity and urease activation might not have correlation.