The Development Of Real-time PCR And CRISPR/Cas12a-based Detection Methods For Streptococcus Suis

Streptococcus suis is a zoonotic pathogen mainly infected by pigs,can cause acute sepsis,meningitis,arthritis,endocarditis and death in pigs,which can infect people through skin wounds.Thus S.suis poses a potential threat to human health and public health safety.At present,laboratories generally use methods such as strain isolation and culture,biochemical identification,serotyping,and conventional PCR for detection,but these methods are time-consuming,laborious,and unable to perform quantitative analysis.In this study,based on rapid nucleic acid extraction technology,PCR,recombinase polymerase amplification(RPA)and Casl2a detection technology,we established real time PCR and RPA-Casl2a detection methods for S.suis in pig synovial fluid,respectively.It provides a new technical means for the detection of the pathogenic bacteria.Multiple pairs of PCR primers were designed and synthesized for the conserved gene gdh of S.suis.Taking the efficiency and specificity of the real time PCR reaction as indicators,the gdh-F#1/gdh-R#1 primers were screened and used in this study.The sample of 100 μL of pig joint fluid was mixed with reagents A and B for rapid nucleic acid extraction to prepare nucleic acid extract.The 50 μL reaction system was needed for conventional PCR,including Taq enzyme 25 μL,1 μL gdh-F#1 primer(10 μmol/L),1 μL gdh-R#1 primer(10 μmol/L),6μL Nuclease-free Water and 2 μL pig synovial fluid nucleic acid extract.The conventional PCR reaction conditions were 95℃ pre-denaturation for 5 min,95℃ denaturation for 30 s,55℃ annealing for 1 min,72℃ extension for 1 min,35 cycles,72℃ extension for 7 minutes.The amplified products were subjected to gel electrophoresis.With the aid of the gel imaging system,the detection results are determined based on the electrophoretic bands.The detection limit of conventional PCR is 4400 cfu.Based on SYBR Green I,a fluorescence quantitative PCR detection method for S.suis was established.The 20 μL reaction system was needed for the optimized real time PCR,including 10 μL 2×Q3-SYBR-qPCR-Master-mix,6 μL Nuclease-free Water,1 μL gdh-F#1 primer(10 μmol/L),1 μL gdh-R#1 primer(10 μmol/L)and 2 μL pig synovial fluid nucleic acid extract.The reaction conditions were pre-denaturation at 95℃ for 60 s,denaturation at 95℃ for 10 s,and annealing at 60℃ for 30 s,40 cycles.The standard curve was established for real-time PCR.The detection limit was 44 cfu.and the amplification reaction was specific.Although real-time PCR is more sensitive than conventional PCR,it requires expensive equipment.And it cannot be detected on site.At the same time,multiple pairs of RPA primers based on the gdh gene were designed and synthesized this study,the RPA detection method of S.suis was established firstly,29.5μL of Rehydration Buffer to the TwistAmp reaction tube(TwistAmp Basic kits),2.5 μL of each of the forward and reverse RPA primers(RPA-Primers#2,10 μmol/L),2.5 μL of 280 mM magnesium acetate solution,5.7 μL of Nuclease-Free Water were added in this reaction,after vortexing and centrifuging,10 μL of the above mixed reaction solution were transfered to a PCR tube,and 1 μL of pig synovial fluid nucleic acid extract was added,and mixing it well.The RPA system was incubated for 20 minutes at 37℃.The amplified products were subjected to gel electrophoresis,and the optimal primer RPA-Primers#2 was selected based on the electrophoretic bands through the gel imaging system.In order to further improve the detection limit of the method,the RPA and Cas12a detection were combined to establish a one-step rapid detection method for S.suis.Based on RPA-Primers#2,guide RNA#2(crRNA#2)that specifically recognizes gdh gene was designed and synthesized.2 μL pig synovial fluid nucleic acid extract and crRNA were added into the RPA system,and incubating at 37℃ for 20 min.Cas12a located on the tube wall was centrifuged to make it enter the reaction system,and incubating at 37℃ for 30 min.After optimization,the optimal concentration of RPA-Primers#2 is 200 nmol/L,crRNA#2 is 40 nmol/L,ssDNA reporter is 800 nmol/L,and Cas12a protein is 60 nmol/L.After optimization,the optimal concentration of RPA-Primers#2 is 200 nmol/L,crRNA#2 is 40 nmol/L,ssDNA reporter is 800 nmol/L Cas12a protein is 60 nmol/L.The detection limit is 44 cfu and the detection time is 50 min,which can meet the needs of on-site diagnosis.