Molecular Detection Of Four Pyogenic Pathogen, Escherichia Coli, Enterococcus Faecalis, Staphylococcus Aureus And Staphylococcus Epidermidis

Invasion and multiplication of pyogenic infection pathogens, such as Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis,can lead to pyogenic infection of the skin, subcutaneous tissue, and deep tissue.It may the abscess of internal organs,even septicopyemia.antibioticresistance of E.coli, E.faecalis, S.aureus and S.epidermidis is serious and contribute to their emergence as leading hospital pathogens.It iscrucial to detect those bacteria, rapidly and accurately, in the clinical diagnosis.Generally,there are three methods, culture method, immuno logical test, and molecular biological methods, used to detect bacterias. At present,it is the gold standard of Clinical bacteria detection for isolated specific pathgen(s). But it is such long time, complex operation and high requirement on staff. In current study, a PCR-based method used to detectE.coli, E.faecalis, S.aureus and S.epidermidiswas established. In addition, a loop-mediated isothermal amplification(LAMP) detectionfor S. epidermidis was developed. All of the four bacterias were analyzed by local BLAST again NCBI nt database in order to screen out the specific genes. The specific genes of E.coli,E.faecalis, S.aureus and S.epidermidis are LafF(GI:12887815), TranR(GI:13347275), PurE (GI:12329975), LysE(GI:3240523) and DeoR(GI:3240916), respectively. Of these, the last two genes are used to detect S.epidermidis. Specific primers were designed to amplify respective genes above-mentioned. The conventional PCR reactions were optimized by adjusting the dose of Mg2+ and renaturation temperature. Then optimizing conditions were selected to evaluate the specificity and sensitivity of this detecting system. The specificity of the assay was evaluated by testing ten bacteria, includingE.coli,E.faecalis, S.aureus, S.epidermidis, Klebsiellapneumoniae, Citrobacterfreundiicomplex, Salmonella typhi,Acinetobacter junii, Staphylococcus haemolyticus and Morganellamorganii. Fifty-eight clinical isolates of those bactria,10 clinical isolates ofE.coli,E.faecalis, S.aureus, S.epidermidisand three strains of other bacteria were detected and showed excellent specificity, i.e. no product was obeserved with any of the other non-target bacteria.This reaction can detect 2 copies/reaction among the four bacterias. Therefore,the sensitivity of this system is very high. we chose the best combination of Mg2+ concentration and renaturation temperature for multiple PCR amplification, according to optimized conditions in the single PCR. The final conditions of reaction are determined as follows:Taq polymerase(5U/μL) 0.3μL, dNTP mix(2.5mM) 4μL, 10×PCR Buffer 5μL, MgCl2(25mM) 3.2μL, the primers(10μM) each of them for 2μL, annealing temperature of the reaction for 57 ℃, extension time for 20 seconds. By the reaction conditions above, we assess the specificity and sensitivity of the multiple PCR. The results are consistent with the single PCR, it can not only amplify the purose bandings, but also attain as low as two reactions.Meanswhile, LAMP to examine DeoR(GI:3240916) gene of S.epidermidis was established. The final conditions of reaction are determined as follows:Bst polymerase(8U/μL) 0.5 μL, 10×Thermpol 2.5μL,MgC12(25mM) 4μL,dNTP mix(2.5mM) 4μL,FIP and BIP each for 2μL,F3 and B3 each for 0.5μL,betaine (1.6mol/L) 2.5μL. This assay could be performed in 75 min at an optimal temperature of 65℃.No ladder pattern was seen with any of the other non-S. epidermidis bacteria. The sensitivity of LAMP was found to be approximately 105 copies per reaction when an incubation period of 60 min was used.In conclusion, a PCR-based method used to detectE.coli, E.faecalis, S.aureus and S.epidermidiswas established and showed excellent specificity and sensitivity. Moreover, LAMP assay targeting the DeoR family transcriptional regulator gene can detect S. epidermidis rapidly and effectively.