Development And Application Of A Multiplex PCR Assay For Detection Of Swine-originated Enterococci Spp.

Enterococci are not only the predominant causes of endocarditis, peritonitis, urethritis and meningitis in hospital patients, but also lead to more and more cases of infection in animals such as swine, sheep, chicken, duck, and so on. Although the members of Enterococcus have increased to forty-one species, the most infectious ones are E. faecalis and E. faecium. Among the pathogenic enterococcal virulence factors, ClyA,AS and Esp are regarded as the most common substances. Specific primers for E. faecalis and E. faecium and those for the cylA,AS,esp virulence genes were designed according to the sequences registered in the GenBank database, and the multiplex PCR methods were set up and then applied in detection of clinical isolates, so as to provide an approach for E. faecalis and E. faecium identification.The Enterococcus genus-specific primers were designed based on the 16S rRNA gene sequence published in the GenBank database, and species-specific primers for E. faecalis and E.faecium were designed on the sodA gene sequence. Taking the two type strains ATCC29212 and ATCC33186 and the five systemically-identified swine-derived enterococcal strains as models, the multiplex PCR conditions were optimized, and were finally determined as 95℃5 min, 95℃40 S, 49℃1 min, 72℃1 min 10s, 30 cycles, and 72℃10min,ending at 4℃. Enterococcus genus-specific and species-specific bands were both observed under the above reaction conditions, while none was discovered with Staphylococcus, Streptococcus or Lactococcus; for the E. sanguinicola bacteria, only the genus-specific bands were acquired. The sensitivity test showed that the minimal detectable dosage was 1 ng of DNA. To further verify the specificity of the amplification, the PCR products were transferred into pTG19-T carrier and were sequenced and and the results showed a high homogeneity with the reference sequences.A multiplex PCR method was plotted for the simultaneous detection of the genus of Enterococcus and the three critical virulence genes cylA, AS and esp. the N9 strains (E. faecalis, positive with cylA, AS and esp genes.) were used as models, and the reaction was optimized as 95℃5 min; 95℃30 s, 56℃1 min, 72℃1 min 10s, 30 cycles, and 72℃10min,ending at 4℃.All the three virulence gene bands were successfully amplified with the model strains, but not with the negative isolates except the genus-specific bands. The sensitivity of this test showed that the minimal detectable dosage was 100 pg of DNA.The suspect Enterococcus isolates from infected pigs, swine feces and raw pig flesh were determined by means of mutiplex PCR method,and the results revealed 190 strains of suspect isolates,158 strains of E. faecalis and E. faecium, with 19 strains of E.spp. unidentified to species level. E. faecalis and E. faecium are the dominant bacteria in three sources of samples. The highest detection rates of E. faecium in the isolates from the infected swine and raw pig flesh were 54.3%和55%, and the detection rate of E. faecalis from swine feces is 46.7%. The highest Virulence gene detection rate of strains from infected pigs, the detection rates of cylA, AS, and esp were 83.3%, 26.4% and 55.4%. The detection rate of cylA gene was high in three kinds of samples from different sources in the detection of virulence gene detection.