Development Of Loop-Mediated Isothermal For RapidDetection Of Streptococcus Suis Type2

With the rapid development of the breeding industry,there are more large-scale farms and intensive breeding.Under the condition of high-density breeding,the immunity of the pig herd decreases and the stress factors increase.As a result,the incidence of opportunistic streptococcus in the upper respiratory tract has been increasing year by year,causing serious economic losses to pig farms.In particular,the newly emerged toxic shock syndrome（STSS）caused by streptococcus type 2 in 1998and 2005 poses a serious threat to the safety of human life.SS2 has strong pathogenicity.If it is found not timely enough,even if a large amount of antibiotics is given in the later stage,it cannot avoid the occurrence of severe neurological sequelae such as paralysis,deafness,blindness and movement disorder,and it can only be eliminated as a residual pig.Because the traditional detection method,the PCR technology,serology,biochemistry and immunology,etc.,most of the time is longer,and higher requirements of instruments and equipment,and streptococcus has 35 serotype,resulting in the traditional method for testing the causing huge consumption of reagents and consumables,and the traditional detection methods,such as PCR,the instrument is generally more expensive price,operation is also tedious,is not conducive to application at the grassroots level.LAMP,LAMP（loop-mediated isothermal amplification）technology can overcome the traditional testing instrument is expensive,time consuming long shortcomings,fast and exact,and testing at the grass-roots level to the popularization and application.According to LAMP principle,two primers were designed by using NCBI to search for conserved sequences of streptococcus suis type 2 virulence factors.Lysozyme release protein(Muramidase.A total of 8 primers were designed in 6regions respectively using the release protein（MRP）virulence factor and the Capsular polysaccharide（CPS 2J）virulence factor as templates.1.Screening of the best reaction systemGradient by setting the single factor respectively to the reaction system of inside and outside primers ratio（F3:FIP,B3:BJP）,concentration of Mg²⁺end concentration,dNTPs is optimized,the introduction and inner primers than gradient of 1:1,1:2 and1:3,1:4,1:5,1:6,Mg²⁺final concentration gradient of 0 mM,1 mM,2 mM,3 mM,4mM,5 mM,dNTPs final concentration gradient of 0 mM,1 mM,2 mM,3 mM,4 mM,5 mM.The optimal reaction system was finally established LAMP:in irfpa MRP virulence factor under the 0.2mM,primers within each 0.6mM,Mg²⁺concentration is2 mM,dNTPs concentration is 3 mM,10 x 2.5μL ThermoPol buffer,1μL Bst DNA Polymerase,template 1μL,filling water to 25μL.Irfpa in CPS2J virulence factor under item 0.1mM,primers within each 0.3 mM,Mg²⁺concentration is 2 mM,dNTPs concentration is 1 mM,10 x 2.5μL ThermoPol buffer,1μL Bst DNA Polymerase,template 1μL,filling water to 25μL2.Optimization of the best reaction conditionsBy setting the single factor gradient respectively optimize the reaction temperature and reaction time,reaction temperature gradient is 60℃,61℃,62℃,63℃,64℃,65℃,66℃,the time gradient for 20 min,30 min,40 min,50 min,60min,70 min.Results show that the MRP virulence factor of primer under 40 min at61℃time can complete amplification.CPS2J virulence factor under the primer under65℃for 30 min time complete amplification.3.LAMP sensitivity testThe SS2 genomic DNA was subjected to 10^-n times gradient dilution,and 10⁰,10^-1,10^-2,10^-3,10^-4,10^-5,10^-6,10^-7,10^-8 times dilution of genomic DNA was used as the template for routine PCR reaction and LAMP reaction,respectively.The results showed that the sensitivity of LAMP method was 100 times higher than that of PCR,and the minimum detection amount was 0.215 pg/L.4.LAMP specific detectionSS2,SS7,SS9,SS14,Salmonella enteriditis,Escherichia coli,Bacillus subtilis,and haemophilus parathophilus as the templates,respectively,for LAMP reaction using the method established in this study to verify the specificity of the method.The results showed that only SS2 could amplify the specific bands,and green fluorescence was observed after adding fluorescent dyes.Other bacteria can not prove that this method has a good specificity.In this study,SS2 LAMP rapid detection method was successfully established,which is simple to operate,highly sensitive,and has high specificity.The experimental results can be directly observed with the naked eye,and it is applicable to rapid diagnosis at the grassroots level and in the field,providing an effective theoretical support for the clinical diagnosis of SS2.