Multiplex-PCR Detection Method And Application For Streptococcus Equi Ssp. Zooepidemicus

Streptococcus equi ssp.zooepidemicus(SEZ),a lancefield group C Streptococcus, infects a wide range of animals,causing meningitis,septicemia,arthritis,endocarditis and sudden death in pigs.In China,SEZ is also the second most important pathogen of swine streptococcal disease,it made substantial economic losses for pig industry and became a substantial threat to human health especially those involved in the pig industry.Fibronectin-binding protein(FNZ) is an important surface protein and virulenceassociated factor of Streptococcus equi ssp.zooepidemicus,which is to mediate substrate adhesion of eukaryotic cells.It is one of the main adhensin of SEZ.The main function of FNZ is to mediate the binding to Fibronectin in extracellular matrix of connective tissue. M-like protein(SzP) is a cell surface-anchored protein that conveys the resistance to phagocytosis.It is also the important virulence factor and protective antigen with opsonin function.Based on the sequence of fnz and szp gene published on the GenBank of Streptococcus equi ssp.zooepidemicus,primers were designed for amplification fnz,szp gene from genomic DNA of Streptococcus equi ssp.zooepidemicus ATCC35246 by polymerase chain reaction(PCR).Recombinant plasmid was constructed by cloning PCR product into the clone vector pMD-19.Also the 540bp and 700bp recombinant fragment was verified by restriction endonuclease analysis and sequencing.The results showed the amplified genes shared more than 99%homology with the fnz gene and szp gene published on GenBank.A multiplex- PCR detection method for Streptococcus equi ssp.zooepidemicus Was constructed based on the superoxide dismutase A encoding gene sodA,fnz gene,szp gene,and was optimized.The results showed the multiplex- PCR detection method shared high specificity and sensitivity.The sodA,fnz and szp gene could be detected in the same time using above multiplex- PCR detection method only in 14 Streptococcus equi ssp. zooepidemicus strains,but there is no the same results in 6 other Streptococcus species strains,and sodA,fnz and szp gene could be amplified from 37 ng/ml genomic DNA of SEZ ATCC35246 strain.An epidemics investigation of swine streptococcosis caused by SEZ was performed using above multiplex- PCR detection method,SEZ strains were detected from milk samples,porck samples and swine throat and nose swab samples.3 SEZ strains were isolated and identificated from the positive samples detected by multiplex- PCR.These results showed the constructed multiplex- PCR detection method for SEZ is helpful in the control of swine streptococcosis.