Development Of Multiplex PCR For Diagnosing The Diseases Caused By Strptococcus And Preparation Of Monoclonal Antibodies Against The Two Virulence Factors Of Streptococcus Suis Sreotype 2

Streptococcus suis and Streptococcus equi subsp.zooepidemicus are the important pathogenies of diseases in pigs. Streptococcus equi subsp. zooepidemicus cause arthritis and septicimia in pigs. And Streptococcus suis serotype 2 is a major cause of meningitis, septicemia, arthritis, and bronchopneumonia in young pigs and can cause meningitis in humans. In many countries ,much attention were pafd to Streptococcus suis serotype 2 and many studies were foucus on virulence factors.The main virulence factors of it were capsular polysaccharide, extracellular protein factor(EF), suilysin, muraminidase-released protein(MRP).On the base of the appearance of MRP or EF, Streptococcus suis serotype 2 could be divided into several types: MRP+EF+ ,MRP-EF-, MRP+EF*,MRP*EF, MRPEF+, MRP+EF- ,MRP-EF* et al.In the epidemiological studies in JIANGSU regin of China, MRP+EF+ was correlate to high virulent strains. And it was very important to diagnose, prevention and curceradication programs the disease caused by Streptococcus suis serotype 2 that detecting the MRP. EFand more studying the function of MRP and EF in the pathogenesis.A multiplex PCR assay was developed for detection the specimens from pigs infected with S. suis strains and (or ) Streptococcus equi subspzooepidemicus. The series of the M-like-gene of Streptococcus equi subspjooepidemicusin and cps2J-gene of Streptococcus suis serotypel were 364bp and 675bp PCR products respectively. The PCR was evaluated using the admixture culture and mixed infection of S. suis strains and Streptococcus equi subsp.zooepidemicus in tonsillar specimens from pigs . The results demonstrated that this multiplex PCR is a highly specific and sensitive diagnostic tool for the detection of pigs carrying S. suis strains and (or) Streptococcus equi subspjzooepidemicus.Another multiplex PCR assay for the detection and identication of MRP and EF of Streptococcus suis serotype 2 strain in tonsillar was developed and evaluated. The series of the mrp-gene, ef-gene and cps2J-gene of Streptococcus suis serotypel were 885bp,433bpand 675bp PCR products respectively. When DNA from the bacteria of 5. agalactiae ,P.pneumotropica and S.eqiusimilis were used as templates for amplification, no product was detected. Compared to bacteriological examination, the multiplex PCR was more sensitive and specific.EF protein and MRP protein extracted from Streptococcus suis serotype 2 strain SS2-H ,the recombinant EF protein were purified by different methods respectively. EF The purification of EF, MRP protein and rEF were immunized BALB/C mice respectively. The spleen cells were fused with the SP2/0 by PEG-1500. The hybridoma sueprunt was detected with indirect ELISA .Two,one ,three monoclonal antibody cell respectively only directing to EF,rEF ,MRP were obtained by diluting several times and ascites was identified with Westen-blot.One of monoclonal antibody cells were purified labelled by FITC. Direct fluorescence coloring to MRP monoclonal antitody to detect the MRP+ or MRP' strains of Streptococcus suis serotype 2.A multiplex PCR assay for the detection and identication of MRP and EF of Streptococcus suis serotype 2 strain in tonsillar were developed and evaluated. The series of the mrp-gene, ef-gene and cps2J-gene of Streptococcus suis serotypel were 885bp,433bp and 675bp PCR products respectively. When DNA from the bacteria of S. agalactiae .P.pneumotropica and S. eqiusimilis were used as templates for amplification, no product was detected indicating specificity of the primers. Compare to bacteriological examination, multiplex PCR was more sensitive and specific than it.