Establishment Of A Rapid Detection Method For Salmonella Gallinarum

Chicken salmonellosis,caused by Salmonella infection,is the most common infectious disease in chickens.According to the symptoms after infection,it could be classified into dysentery,typhoid and paratyphoid.Chicken salmonellosis has caused huge economic losses to poultry industry and seriously threatened the development of poultry industry.In addition,salmonella has long been the top food safety problem in the world,especially in developing countries.Therefore,it is of great essential to establish a Salmonella detection method which has rapid,specific and highly sensitive.In recent years,nucleic acid isothermal amplification technology has been used in the pathogenic microorganisms’ rapid detection due to its advantages of rapid,simple,sensitive and low cost.This test was mainly carried out three kinds of rapid detection methods of Salmonella Gallinarum,and obtained the following results:1.By reviewing the literature and NCBI database comparison analysis,invA gene(GenBank ID:U43273.1)was choosn and the highly conserved sequence of this gene was select.Use primer explorer V5(https://primerexplorer.jp/lampv5/index.html)and PrimerPrimier 5 software designed LAMP,RPA and SRCA multiple sets of primers,filtered,each method resulted in an optimal set of primers.2.Through optimizing LAMP reaction conditions and reaction system,a visual LAMP method was established for the detection of Salmonella Gallinarum.The optimal LAMP reaction conditions were reacted at 61℃ for 30 min.By optimizing the reaction conditions and reaction system of RPA and combining with LFD strip,a visual RPA-LFD method was established for the detection of Salmonella gallinae.The optimal reaction condition was react at 35℃ for 14 min.By optimizing the reaction conditions and reaction system of SRCA,a visual SRCA method was established for the detection of Salmonella gallinarum.The optimal reaction temperature was 61℃,and the optimal reaction time was 30 min.3.The established visual LAMP method,RPA-LFD method and visual SRCA method can specifically detect the target bacterial strains,and there is no cross reaction with other uncorrelated strains.The lowest detection limits were 2.7×101 copies/μl,2.7×102copies/μl and 2.7×101 copies/μl.which were 100 times higher than those of PCR methods.4.A total of 326 eggs were collected from a large supermarket and market in Yinchuan,Ningxia.Three established methods metioned above were used to detect clinical eggs,and the results were compared with the spread plate results and PCR results.The results showed that the positive detection rate of PCR method was 12.58%,compared with 6.13%of plate coating method.The positive rate of visual LAMP method was 16.56%.The positive rate of RPA-LFD method was 20.55%.The positive rate of visualization SRCA method was 18.71%.The results showed that the established visual LAMP method,RPA-LFD method and visual SRCA method had good specificity and sensitivity,and the detection results were visualized and timesaving,which could realize the rapid detection of Salmonella Gallinarum.By comparing the detection cost,detection time and detection accuracy,we are more inclined to the promotion of visual LAMP method in grass-roots field detection.