Establish RT-PCR Detection Method STY And The SPA

【Objective】To develop Real-Time fluorescent quantitative polymerase chain reaction method todetect Salmonella Typhi and Salmonella enterica serovars paratyphi A. including:(1) Todevelop the Real-Time fluorescent quantitative polymerase chain reaction (RT-qPCR)method to detect Salmonella Typhi.(2) To develop Real-Time fluorescent quantitativepolymerase chain reaction method to detect Salmonella enterica serovars paratyphiA.Combine the optimized simplex real-time PCR to form duplex real-time PCR system,and the detection limit of duplex real-time PCR were evaluated.【Methods】The real-time detection methods to detect Salmonella Typhi and Salmonella entericaserovars paratyphi A were established according to the following protocols:(1) The specific genes were selected by referring to literature and the conservedsequence of the gene, from which the primers and probes for TaqMan real-time PCR weredesigned with the help of Beacon Designer7.0, were got by sequence alignment.(2) Optimize the concentration of primers and probe, whose concentration ranges from100－1000nmol/L and100－500nmol/L respectively, with One-Way ANOVA.(3)The specificity and sensitivity of RT-qPCR were evaluated.(4)To develop RT-qPCR method to detect Salmonella Typhi pathogen in stool samples.(5)Evaluate the sensitivity and specificity of RT-qPCR.(6)Evaluate the sensitivity and specificity of duplex real-time PCR.【Results】（1）Total44S. Typhi isolates were amplified positive by RT-PCR. Other non-salmonella isolates including5dominant non-salmonella enteric diarrheal pathogens andeight species of non-enteric bacteria causing bacteremia with fever symptom were detectednegative. For purified total DNA from pure culture isolates the detection limit of RT-PCRwas500fg per reaction, equal to97copies per reaction. The limit of detection achieved104cfu/g and50cfu/g with crude nucleotide from simulated stool before and afterenrichment, respectively. （2）In the optimized reaction system of Salmonella Typh real-time PCR.The sensitivityand specificity of this reaction system are both100%. The detection limits of the optimizedreaction system to two plasmids are both1.0×102copies/μl, and the amplificationefficiency is90％respectively.(3) In the optimized reaction system of Salmonella enterica serovars paratyphi A real-time PCR. For purified total DNA from pure culture isolates the detection limit of RT-PCRwas100fg per reaction, equal to19.4copies per reaction. The limit of detection achieved104cfu/g and50cfu/g, and the amplification efficiency is90％respectively.