The Stablishment Of PCR Diagnosis Method For Muskra Salmonella Enteritidis

Salmonella Enteritidis was a kind of invading pathogenic bacterium,which had no host specificity,the host included human and many kinds of animals.Not only could Salmonella Enteritidis bring severe economic loss caused by the death of poultry,but also could the pollutional poultry as the carrier of Salmonella Enteritidis do harm to the health of human.It was reported that 40%to 80%food-poisoning events were caused by poultry Salmonella in developed countries such as Japan and USA,the main pathogeny was Salmonella Enteritidis.At present,the study for muskrat Salmonella Enteritidis was less in our country,the main detective methods were isolated culture,biochemistry reaction and serological identification,and the detection procedure was very tedious.Therefore,it was essential to establish a kind of fast,sensitive and specific molecular biology diagnosis method,in order to have early diagnosis and prevention of muskrat Salmonella Enteritidis.This research designed a couple of specific primers based on reported invA gene sequences of Salmonella Enteritidis with Oligo6.0 and Primer Premier 5.0 software,the length of 475bp specific gene fragments were amplificated,and cloning to pGEM-T Easy vector,then enzyme cutting analysis and PCR identification having been done,taking the recombinant plasmid of positive clones to have nucleotide sequence analysis and homology comparison The results showed that the homology was 98.7%between isolated strains gene order of muskrat Salmonella Enteritidis and the gene order of Salmonella Enteritidis reported on GenBank.The results of specific and sensitive experiments showed that there was no cross reactivity among Pasteurella,E.coli and Klebsiella;the least concentration of Salmonella Enteritidis could be detected was 27.46fg/ul,this method could be used for early diagnosis,silent infection,and epidemiological investigation.The PCR diagnosis method established was used to detect fifteen samples,the positive rate was 58%,but the common isolated identification rate was 26%.