Development Of A Multiplex FQ-PCR Assay For The Specific Detection And Quantification Of Bifidobacterium And Bacillus And The Kinetics Of The Intestinal Microflora In Duckling Infected With Salmonella Enteritidis

A new multiplex FQ-PCR assay was developed to target the key commensal bacterials Bifidobacterium and Bacillus in intestinal tract.Real-Time Fluorescence Quantitative PCR (FQ-PCR)was used to enumerate the dynamics change of Bifidobacterium spp,Bacillus spp,Enterococcus spp,Eschericha spp,Staphylococcus spp and Lactobacillu spp in intestinal tract,including duodenum,jejunum,ileum,cecum,rectum from Salmonella Enteritidis(SE)infected and healthy ducklings.1.A new multiplex real time PCR assay was developed to target the key commensal bacterials Bifidobacterium and Bacillus in intestinal tract.The sensitivity assays were able to detect 9 CFU/ml and 6 CFU/ml per PCR reaction.The specificity of the multiplex assay were determined to be 100%specific with the two strains,and related intestinal tract specimens.2.After SE infection,the kinetics of the intestinal microbes showed that:(1)Comparison with the infected was a significantly lower colonization for the Bifidobacterium in intestinal tract.The results showed decrease of the population of Bifidobacterium spp.from hypodermical infection ducklings during 12 h- 6d PI(P＜0.01). There were significant decrease in the Bifidobacterium spp gene copies from nasal-infected samples during 24 h- 9d(P＜0.05);after SE infection orally,a significantly lower colonization for the Bifidobacterium in intestinal tract during 12 h- 9 d(P＜0.05);at late infection,the Bifidobacterium spp.had a significantly increase in duodenum(P＜0.05).(2)There was no significant difference between the infected and healthy ducklings, due to the low detection of Bacillus in the intestinal tract.In jejunal and ileal samples,there was lower positive rate during 4-8h and 24h -6d after hypodermical infection,compared with the healthy ducklings.In duodenal samples,positive rate was 7/12 from healthy duckling,and 1/11 from hypodermical infection duckling.The number of the genus from infected ducklings still lower than the control,in duodenal and caecal samples the positive rates were 7/12 and 8/12 from healthy ducklings,after nasal infection the positive rates were 4/12 and 1/12.In caecal samples,positive rate was 8/12 from healthy duckling,and 3/12 from oral infection duckling.There were significant decrease in the Bacillus spp.gene copies from jejunal samples at 36 h(P＜0.01).(3)After SE infection,the duodenal samples showed first increase and then decrease of the population of Enterococcus,especially during 4-36h(P＜0.05)after hypodermical infection,during 12-36h after oral infection,during 4-36h after nasal infection,reached the peak at 24h(P＜0.01),12h(P＜0.01),24h(P＜0.01);decreased significantly at 48h after hypodermical infection(P＜0.05),then re-grow to normal level;there were significant increase of the genus from rectal samples of infected ducklings,during 4-48h(P＜0.05) after hypodermical infection,at 8-36h(P＜0.05)after nasal infection,during 24-36h(P＜0.05)after oral infection.(4)In ileal and caecal samples,there were significant increase during 8-48h after hypodermical infection(P＜0.05).The genus of Eschericha still showed a greater tendency in the duodenal samples during the 4h to 8h,and 36h after nasal infection(P＜0.05),and in the jejunal samples at 4h(P＜0.05),8h(P＜0.05),36h(P＜0.05)and 72h(P＜0.01)PI.At 48h PI,a significant difference(P＜0.05)of Eschericha spp.was observed,and there was an increase in the number of Eschericha spp.from 8h to 72h in caecal samples(P＜0.05).(5)After nasal and hypodermical infection,the samples showed increase of the population of Staphylococcus spp.,especially during 4-8h,24-36h after hypodermical infection,have a peak value at 4h PI(P＜0.05)and 24h PI(P＜0.05);and during 24-72h after nasal infection;there were two least at 24h(P＜0.05)and 6d(P＜0.05)in the duodenal samples after oral infection,and increase at 8h(P＜0.05)in the jejunal samples;the ileal and caecal samples showed significantly decrease at 4h,then increase at 8-36h.(6)There were significant decrease in the Lactobacillu spp.gene copies from ileal and caecal samples,especially during 8h-6d after hypodermical infection(P＜0.01);during 24h-9d after nasal infection(P＜0.01);during 8h-9d after oral infection(P＜0.01).In caecal samples,there were significant decrease during 12h-9d after oral infection(P＜0.05).