Establishment Of A Rapid Detection Method For Salmonella Indiana

Salmonella spp.are important zoonotic pathogens.According to statistics from the US CDC,in the United States,Salmonella causes about 1 million illnesses,19,000 hospitalizations,and 380 death.In China,about 70%of bacterial diseases were caused by Salmonella.Salmonella spp.contain a variety of serotypes,but the prevalent serotypes are Salmonella typhimurium and Salmonella enteritidis.However,Salmonella Indiana has become prevalent in broiler farms in China among multiple serotypes in recent years.Since the first reported in 1984 in China,Salmonella Indiana has been widely prevalent in animal,food,and breeding environment.In addition,Salmonella Indiana?S.Indiana?shows multiple drug resistance,which makes more difficult to prevention and control it.This pathogen has become an important serotype in many fields such as poultry disease prevention,food safety,and public health.Loop-mediated isothermal amplification?LAMP?is an emerging nucleic acid amplification technology invented by Notomi.This technology designs primers for the six regions on the target,using DNA polymerase with strand displacement activity to amplify a few of copies to millions of times in an isothermal environment within 1 hour.LAMP avoids temperature changing steps in polymerase chain reaction?PCR?.LAMP assay has received widespread attention and has been used in many fields such as disease diagnosis.In this study,primers were designed by screening specific sequences of S.Indiana for establishment of LAMP,providing an effective technical means for the scientific prevention and control of S.Indiana.Main findings are as follows:?1?Establishment of LAMP assay:In this study,a set of primers were designed based on S.Indiana specific sequences.LAMP assay were optimized according to different reaction conditions.At last,25?L reaction system including 1?L of Bst DNA polymerase,3?L of dNTPs,2?L of Mg²⁺,1?L of F3/B3 primers,0.75?L of FIP/BIP primers,2?L of template,2.5?L of Buffer and sterilized double distilled water to make up the system.The reaction conditions were isothermal amplification at 64?for50 min,and inactivation at 80?for 10 min.As a result,the amplification products could be digested by restriction enzyme as expected showed that LAMP of S.Indiana has been successfully established.?2?Identification of specificity and sensibility:41 reference strains such as S.Indiana were used to verify the specificity of this method.The results showed that it had a good specificity with S.Indiana were positive.Plasmid containing the target sequence was detected by ultra-micro spectrophotometer to calculate the copy of the plasmid and make a tenfold dilution to detect the detection sensitivity of LAMP.The detection limit of the LAMP assay was 5.9×10¹ copies/tube better than conventional PCR?5.9×10² copies/tube?.?3?Establishment and application of LAMP visual response:Calcein was used as an indicator to establish a visual detection method.For 359 samples isolated from clinic,a total of 65 samples were positive detected by LAMP visual response,while 64 were positive through the bacteriological method.