Establishment Of Routine Pcr And Real-Time Fluorescence Quantitative PCR For Detection Of Salmonella Pullorum

Pullorosis caused by Salmonella pullorum is one of the bacterial diseases which endanger poultry industry,and it seriously affects the healthy development of China's poultry breeding.pullorosis has been listed as the bacterial disease that must be purified by the government.At present,the most effective prevention and control measure is to strengthen the quarantine of chickens and to timely weed out the infected chickens,to gradually achieve the purpose of purifying the poultry.Therefore,the establishment of a rapid and accurate detection method is of great significance for purification of pullorosis.In this study,a comprehensive bio-informatics analysis of the whole genome of S.pullorum which number is ATCC9120 registered in Gen Bank is carried out.Through comparing it with the other genomes in NCBI database,the study screened out the conserved sequence of SEEP9120₀17695,which is a fragment of the genome of S.pullorum.This gene is highly specific and is identified as the target gene for the detection of S.pullorum.According to the selected target gene,11 pairs of candidate primers were designed.The study adopted 33 strains of Salmonella and 11 strains of non-Salmonella,to verify the specificity of each pair of primers by PCR method.Finally,a pair of primers with the best specificity was selected,and the amplification?219 bp?is suitable for the real-time fluorescence quantitative PCR method.Besides,the study established a routine PCR detection method after the optimizing of the PCR conditions.The sensitivity of the established method is up to 213 pg/?L when the genome of S.pullorum was detected.The sensitivity of the pure culture is 2.1×104 cfu/m L.When the samples were detected for the enrichment of the chicken manure,eggs and chicken by this method,for the mildly contaminated samples?13 cfu/10 g samples?,the S.pullorum was detected after 6 to 8 hours of culture.The sample of severely contaminated?13×107 cfu/10 g sample?can be detected after 2 hours of culture.But in the detection of egg samples,it needs 1 to 2 more hours for enrichment and then the positive samples can be effectively detected.According to the reaction conditions of routine PCR method,real-time fluorescence quantitative PCR method was established.According to the 44 strains of the amplification curve and dissociation curve among the experimental results of specificity,the analysis shows that it has the best specificity when the method is used for detecting S.pullorum.According to the established real-time fluorescence quantitative PCR method,the sensitivity of the genome of S.pullorum is 34 fg/?L.The study polluted the chicken manure,eggs and chicken with S.pullorum and established the artificially contaminated samples.At the same time,thereal-time fluorescence quantitative PCR method and standard method for isolation and identification of bacterial were used to detect the contaminated samples and evaluated the effect of this method.When the initial level of the contamination of S.pullorum is as low as 2 cfu/10 g in chicken manure or chicken,the positive rate of real-time PCR method is 24/27?88.9%?after 6 hours of culture.The result is completely consistent with that of the standard method.When the initial level of the contamination of S.pullorum is as low as 2 cfu/10 g in chicken manure or chicken,the positive rate of real-time PCR method is 21/27?77.8%?after 6 hours of culture.The result is consistent with that of the standard method by 91.7%.The study screened out a specific gene of S.pullorum as the target gene for detection,and established routine PCR and real-time fluorescence quantitative PCR detection methods.There are their own advantages of two methods in the actual detection.Routine PCR detection method is simple and economic.The sensitivity of real-time fluorescence quantitative PCR detection method is higher and the detection is more accurate.We can choose one of the two methods according to the actual situation in the detection.The study provides poultry breeding with the technological means for the rapid diagnosis of pullorosis and also to provide a basis for the future identification and detection of S.pullorum in laboratory.