Detection Of Salmonella In Secondary Effluent By PCR

The reclamation and reuse of wastewater is an important strategic decision to ease water shortage in China. After secondary treatment, water quality of sewage has been improved, but the absolute number of bacteria in it is still considerable, and in those bacteria,there is a variety of intestinal pathogens. Salmonella is one of the most typical intestinal pathogens. It can cause quite a few human diseases, such as typhoid fever, acute gastroenteritis,bacteremia and septicemia. Among those diseases, gastroenteritis is the most common one, the symptons of which are chill-fearing and fever, accompanied by headache, dizziness, nausea, vomiting, abdominal pain, diarrhea, In severe cases, dehydration and electrolyte imbalance can be seen. In view of this, the detection of Salmonella in urban wastewater reclamation and reuse, through which the presence of it can be known, is practically significant to the improvement of the sewage treatment process for removing pathogens and the evaluation of the risks of pathogens.This study selected membrane adsorption-elution as sample pre-treatment method for secondary effluent, and considered the impact of membrane material, membrane pore size and the type of eluant on the recovery of salmonella from secondary effluent, and then the best membrane adsorption - elution method was established. the concentrated Salmonella was detected using the selected primers 139 and 141. The optimum condition for the PCR detection was obtained by orthogonal experiments regarding five factors at four levels.Based on the qualitative PCR method, the quantitative PCR method for detection of Salmonella was established. This study also used recombinant plasmid and the genomic DNA of standard strain as templates to create standard curves, respectively, and then the two kinds of standard curve were compared. Furthermore, we detected the secondary effluent consecutively by PCR, and made an analysis of the distrbution of Salmonella present in secondary effluent and the relationship between distrbution and several other factors.The following conclusions were obtained:(1) cellulose acetate(ester)membrane filter has the best effect on the concentration of Salmonella in the four common filters. Compared with the 0.45μm membrane, 0.22μm membrane can reject more bacteria.3% beef extract solution works well as the eluant of Salmonella.Glycine buffer is an excellent eluant of virus, but its performance in eluanting bacteria was not good.(2) The optimized PCR reaction system was established: the total reaction volume was 25μL,0.5μL template,0.4μmol/L primers,1U Taq enzyme,800μmmol/L dNTPs, 2.5mmol/L Mg2+.65℃was finally selected as the annealing temperature. The sensitivity of PCR for Salmonella was 0.314 ng/L, and the sensitivity of the whole detection process containing membrane adsorption-elution concentration was 2.13 CFU/mL water sample.The method established in this study showed its advantage over the conventional MPN method in its higher sensitivity and efficiency.(3) The standard curve created by recombinant plasmid was better than that by genomic DNA,as its correlation coefficient can reach 99.9% and good linear relationship. Q-PCR has a nice specificity, and The sensitivity of it was 15.6 GEC/μL.The intra-assay and inter-assay CV of CT was less than 1% and 3%, respectively,which showed the Q-PCR has a higher precision.(4) The secondary effluent has a high positive rate and Salmonella concentration. The average concentration reached 3.57×104GEC/L, but had no significant change regulation with the time,which may be caused by the limited number of samples. The monitoring result showed that Salmonella does not correlate with fecal coliform and fecal streptococcus, and has no significant correlation with pH and turbidity but a significant correlation with water temperature (p=0.049<0.05),the correlation coefficient of which was 0.65.This was also in accordance with the increasing trend of Salmonella concentration as weather gets warmer.