Quantitative PCR Detection Method Of Salmonella Typhi In Secondary Effluent

Global freshwater resources are facing two major problems: water pollution and water shortage. Wastewater reuse in an certain applicable scope,provides us with an economical and reliable resources,and can save high-quality source of drinking water. At present,the urban sewage has become a stable reclaimed water. Besides,the amount of reclamation is also on increasing. After secondary treatment,a variety of pathogenic microorganisms would decline. But there were still some pollutants such as bacteria,viruses , microorganisms , the solubility of influencing recycle which can not completely removed by secondary treatment. The Salmonella typhi is one of the most typical intestinal pathogens,widely distributed in the water. Salmonella typhi is the main pathogen of causing cholera,typhoid and other intestinal diseases and food poisoning. In response,establishing a rapid,specific and sensitive detection of Salmonella typhi is urgently needed. This is practically significant to the improvement of the sewage treatment process for removing pathogens and the evaluation of the risks of pathogens.In order to establish a method for detection of the Salmonella typhi in secondary effluent by real-time fluorescent quantitative polymerase chain reaction (QPCR) technique,this study tried to design and synthesize a pair of coverage and specificity primers ST3 and ST4 for Salmonella typhi. Using membrane adsorption-elution method to concentrate Salmonella typhi,and analyzing the facors impact of Salmonella typhi recovery. The effect of applying qualitative filter paper with different pore sizes to improve the recovery of Salmonella typhi was investigated. Finally decided the best way to pre-treatment. Meanwhile,we detected the secondary effluent consecutively by QPCR and analyzed the test results.The detection results showed that this method can only detect specific amplification fluorescent signal from those water which contained Salmonella typhi. But not reacted with other bacteria's DNA. Fully demonstrated the high specificity and sensitivity of detecting Salmonella typhi.Based on the QPCR method to detect Salmonella typhi recombinant plasmid,The amount of template ranges from 2.8×10~1～2.8×10~8 GEC have a good linear relation (R=0.9955). The detection limit was 2.8×10~1 GEC/PCR reaction. The whole processes including extracted DNA from samples and reported the results could not more than four hours.The method of this study had obvious advantages in detecting effect and efficiency. So it was suit for quantitative detection of Salmonella typhi in secondary effluent. As the QPCR technique continues to develop and improve,it will produces more influence in environmental microbial field.