| Elizabethkingia miricola,an emerging opportunistic pathogen,was documented to cause sepsis and urinary tract infection in infants and immunodeficient patients.Recently,it was reported to cause frogs to torticollis,cataracts,visceral hemorrhage and other symptoms,and it can cause explosive death in a short time.The E.miricola infection caused huge economic losses to China's frog breeding industry.The bacteria can also be a potential zoonotic bacterium,which has a potential threat to human health.Therefore,it is essential to develop a rapid and reliable diagnostic method for the diagnostic of E.miricola infections.In the present study,a duplex PCR method and a real-time PCR were developed for the rapid and sensitive detection of E.miricola.This method could be useful for early detection and epidemiological surveillance of E.miricola.1.Establishment of the duplex PCR method:Based on the sequence alignment,two specific primers were screened targeting on PNGase F gene of Elizabethkingia,ure G gene of E.miricola,which generates two specific amplification of 1192 bp and 246 bp,respectively.Optimization of conditions and tested the sensitivity and specificity of the assay.The result indicated that duplex PCR has good specificity and sensitivity.PNGase F primers only specifically amplify Elizabethkingia spp.,while ure G gene primers can only specifically amplify Elizabethkingia miricola.The detection limit of the assay was4.76×10-2ng/?L.2.Establishment of the real-time PCR method:Based on the whole genome of E.miricola FL160902?Gen Bank accession no.CP040516?,100 house-keeping genes were randomly selected for the primer design.These genes were aligned against the genomic sequences of Elizabethkingia spp.and other bacterial species using the BLASTn program of the basic local alignment search tool.Twenty-seven fragments were selected for the primer design on the principle of keeping genes with lower similarity as much as possible.The primer sets were designed to be specific to E.miricola from the 27 screened genes using Oligo 7 software and then evaluated by PCR.After agarose gel analysis,the specific primers,designed on gene mut T,was ultimately chosen for real-time PCR amplification.The study adopted 5 E.miricola and 13 non-E.miricola strains,to verify the specificity of the real-time PCR.Simultaneously,the sensitivity and reproducibility of the assay were tested.The result indicated that the real-time PCR has good specificity.The five E.miricola appeared an S-type amplification.All non-target bacterial species were either not amplified or observed with changes in fluorescence,but the Cq values were>35.The detection limit of the real-time assay was 145 copies/?L of plasmid and3.49×10-4ng/?L of genomic DNA,which was 100 times more sensitive than conventional PCR.To evaluate the reproducibility of the assay,109to 103dilution series were performed repeatedly to calculate the coefficient of variation?CV?of the Cq value.The intra-and inter-assay CVs for Cq values ranged from 0.25%to 2.54%,indicating good reproducibility.3.Application of the duplex PCR and real-time PCR:The 66 isolates from torticollis frogs were tested with duplex PCR,and some strains were randomly selected for 16S r DNA sequencing to verify the results of duplex PCR.The results showed that of the 66strains,63 strains specifically amplified PNGase F and ure G genes.The five randomly selected positive strains were proved to be E.miricola by 16S r DNA sequencing,while three negative strains were confirmed to be other species.The 27 frogs?54 samples?collected from four farms with a history of severe outbreaks,were tested with real-time PCR and bacteriological culture,simultaneously.The result indicated that 52 samples were positive for E.miricola in real-time PCR,but only 35 samples were positive for bacteriological culture.All samples negative for E.miricola in real-time PCR showed negative results in bacteriological culture.No samples of bacteriological culture positive showed negative in real-time PCR. |
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