Development And Application Of The Liquid Phase Chips Of Streptococcus Suis Advantage Serotype Typing And Main Virulence Factors Screening

The methods of Streptococcus suis serotype just be serum agglutination or synergistic agglutination,but this methods have some shortcomings,for examples,it consume a lot of time and the results difficult to judge when screening a large number of clinical samples.And the introdution of Streptococcus suis serum will spend Two hundred thousand.There are the serotype 1(14),2(1/2),7,9type of detection of Molecular,with the complexity of the epidemic and the further of research,which is only 6 serotypes detection methods can not meet the needs,and the research of virulence factor screening is limited to mutiplex PCR,and can not complete to screen in a system.This paper designed a liquid phase chips of Streptococcus suis advantage serotype typing and main virulence factors screening,details are as follows:1.Development of the liquid phase chips of Streptococcus suis advantage serotype typing and main virulence factors screeningAt first,this study designed and optimized primers of 20 serotype and 7 virulence factors,and divided into six groups of multiplex PCR system under the condition of specificity and no dimers.the first set of serotype is1,2,1/2,3,7,9,14;the second group serotype is 5,8,12,19,25;third group serotype is 21,27,28,30;the fourth group is the serotype 13,15,23,31;fifth group is fbps,gdh,orf2,mrp;sixth group is sly,gapdh,epf.Then optimizated the primer concentration ang annealing temperature between the groups,eventually,six groups of multiplex PCR will be completed in two times detection systems,respectively,the first,second,third,fourth groups,and the fifth,six groups.Appending a TAG sequence to the eraly stage of the specific up-primers'5',use 12 carbon bridge as a termination signal in the between of its,appending a Biotin to the specific down-primers' 5',choices the microspheres that have the relatively comolemented of the TAG sequence(with anti-TAG sequence),while setting six hybridization temperature gradient optimization,select the optimum annealing temperature of 40 ? as liquid chip according to the results.Finally,taking the test for strptococcus suis serotype classification and main virulence factor screening of liquid phase chips.By sensitivity,specificity and reproducibility of the test to verify the performance of liquid-chip,there are no cross-reactivity of dispaly each probe specificity primer combinations with other bacteria;and a minimum sensitivity of detection was lpg/?L,more than 100 times conventional PCR of sensitivity(105pg/?L);a repetition rate of 98%,greater than 95%.good reproducibility.2.Development and application of the liquid phase chips of Streptococcus suis advantage serotype typing and main virulence factors screeningUsing PCR methods and MS technology from 350 clinical samples,we screened 60 Streptococcus suis,then this study design Luminex taking serotype typing virulence factors screening tests,and the results were verified with standard serum(serum agglutination)and gene sequencing.The test completed the 58 serotypes of S.suis typing with detected serotypes serotypes 2,3,5,7,9,21,27,and another two suis no stereotypes.Wherein the detection rate is the highest serotype 7,9,accounting for 26.7%,26.7%,21.7%.Virulence factor screening results show:there are 21 strains containing both virulence factor mrp,epf,sly three kinds of virulence factors,the percentage of 35%.The results of serum agglutination and gene sequencing of detect positive coincidence rate was 96.7%and 100%,respectively.The test to establish liquid chip technology of serotype typing and main virulence factors screening with high speed,high throughput,and high specificity and accuracy.The quadruplex xTAG methods had established successfully in this study for detecting twenty-seven(20 S.s serotype and 7 virulence factor)rapidly at the same time.This detection method build a new,rapid and high-throughput technology platform for the serotype typing and virulence factors screening,it also laid the foundation for application of xTAG technology in erotype typing and virulence factors screening of other bacteria.