Multiple PCR Detection Establisment And Optimazation Of Lmonella With Virulence Plasmids

Salmonella is a common and important infection pathogen of human and animals.It could cause animal many diseases,such as chicken typhoid,pig paratyphoid,misbirth et al,and also cause human many diseases,like typhoid,paratyphoid,blood poisoning,gastroenteritis,food poisoning,local purulent and so on.Salmonella virulence plasmids of different molecular weights have been identified from S.typhimurium,S.choleraesuis,S.dulin,S.enteritidis,S.abortusovis,S.gallinarum.and S.pullorum.It has also been reported that virulence plasmids exist in salmonella typhi.The toxicity owned by Salmonella strain with virulence plasmids is stronger from a few hundreds to tens of thousands times than those with non-virulence plasmids.PCR technology has become a powerful tool for disease investigation and pathogen identity due to its simplicity,ease of use,high sensitivity and strong specificity.Multiple PCR technology has currently been employed primarily in detection of multiple pathogenic microorganism because it has many advantages such as high efficiency and productivity,low cost,fast speed.The establishment of multiple fluorescence quantitative PCR detection system combining multiple PCR with real time PCR has become a research direction of microorganism especially in pathogenic microorganism detection.Here we used molecular biology and bioinformatics tools for gene search,sequence blast,target sequence screen and multiple PCR primer design.Homology blast was carried out for conserved operon of histidine transferase(hisJ)in Salmonella genus and H1 antigen H1-c and H1-i sequence of conservative district and positive regulated gene(spvR)of relative virulence factor in Salmonella virulence plasmids.Then specific primers for Salmonella genus and Salmonella virulence plasmids were designed for multiple PCR using Primer Premier 5 software.Multiple PCR examination was performed for common Salmonella with virulence plasmid according to above primers.In this study,we chose common Salmonella standard strain with virulence plasmid and common non-Salmonella standard strain,employed step-by-step strategy,optimized single,douplex and multiple PCR amplification conditions and testified primer specificity gradually.Experimental results show that using hisJ gene primer salmonella amplification out 359 each bp and 93 bp target gene;Using the spvR gene primers with salmonella virulence plasmid of amplification of 789bp and 293 bp target gene;Using the H1-c antigen specificity primer amplification of 623 bp and 200 bp target gene;Using the H1-I antigen specificity primer amplification of 537 and 143 bp target genes.Our results showed that special amplification by triple PCR was able to be carried out in S.typhimurium,S.aberdeen,S.choleraesuis and to be supplemental evidence for serotype identification.The PCR program for this study:94℃ starting degeneration 5 min,94℃ modified 45 s,52℃ annealing 45 s,extension 72℃ for 1 min,amplification of 32 cycle,the last cycle at 72℃ for 10 min.Multiple PCR system of 30μL,this research to the result of the multiple PCR system optimize annealing temperature 52 ℃,primers(10μmol/L)to add the amount of the optimization results of 0.8μL,Mg2+(30mmol/L)in the concentration of the optimization results of 2.0μL,dNTPs(2.5mmol/L)concentration of the optimization results of 0.8 μL,Taq polymerase(5.0 U/μL)optimization results of 0.4μL;Primers sensitivity test results show that the system can detect the minimum concentration of DNA template for 100 pg;The artificial infection test results show that the system detection specificity is very strong.The purpose of this research for small fragments can amplify the gene primer,step by step,the single PCR,double PCR,multiple PCR,validation results show that the primer in the amplification of salmonella has specificity,nothing but specific amplification,without formation of primer dimers,accord with the requirement of multiple PCR to do real-time quantitative.Multiple fluorescence quantitative PCR detection kit could be developed according to our research combined with real time fluorescence quantitative PCR.It would facilitate rapid quantitative detection of infection of Salmonella and Salmonella with strong plasmids virulence in samples.This technology would be of much practical significance in public sanitation,food sanitation,stockbreeding and customs quarantine.