Development Of Multiplex PCR Kit For Detection Of Streptococcus Suis

Streptococcus suis is one of the most important swine pathogen worldwide,belonging to Gram-positive bacterium and is responsible for massive economic losses to the swine industry.The infection of S.suis could lead to severe respiratory tract diseases and systemic diseases,including arthritis,pneumonia,septicaemia and meningitis.As a zoonotic pathogen,SS2 is also a major threat to public health.In China,outbreaks of human-associated infection occurred in Sichuan province in 2005 and in Jiangsu province in 1998.Currently,at least 29 true S.suis serotypes have been identified.Streptococcus suis serotype 2(SS2)is considered the most virulent serotype and is the serotype most frequently associated with disease.Research on the virulence of SS2 provide a better understanding of the SS2 infection mechanism and contribute to the prevention and treatment of streptococcosis.1 According to the previous results about the spectral analysis of 101 SS2 strains' virulence gene clusters,the dominant virulence gene were selected as the target.On the basis of virulence-related sequence literatured and published in GenBank,3 primers were designed and synthesized to establish a multiple PCR assay.The PCR,intending to detect the 3 major virulence-associated factors epf,endoD,permease and forecast SS2's virulence was developed by combination and optimization.Results showed that the method was highly specific,and the sensitivity of multiple PCR reached 104CFU/mL.Finally,a detection kit was established on the basis of the developed Multi-PCR assay.The reproducibility of the detection kit is good.The efficiency of the kit was stable after being stored at-20°C for six months.2 Refer to the relevant serotyping genes sequence(gdh and ChzM)literatured and published in GeneBank,the two gene were synthesized to establish a multiple PCR assay.The PCR,intending to detect the 2 typing genes gdh and ChzM and the now sterotye Streptococcus suis was developed by combination and optimization.Results showed that the method was highly specific,and the sensitivity of multiple PCR reached 105 CFU/mL.Finally,a detection kit was established on the basis of the developed Multi-PCR assay.The reproducibility of the detection kit is good.The efficiency of the kit was stable after being stored at-20°C for four months.3 Bacteriocin is a kind of antimicrobial secretory proteins which produced by a variety of microotganisms,although there are few reports on the bacteriocins of Streptococcus suis.In this study,27 of the 530 Streptococcus suis exhibited antibacterial activity against pathogenic Gram-positive isolates.Two bacteriocin-related structural genes(scnA and nisinA)in these 27 strains were detected by PCR,showing the prevalence of scnA and nisinA was 33.3%and 14.8%respectively.Streptococcus suis CZJYW024 exhibited strongest antibacterial activities.Consequently,the aim of this study was to characterize the bacteriocin produced by S.suis CZJYW024,with a view to a potential therapeutic and preventive application.The inhibitory substances produced by this strain were purified primarily,whose inhibitory zones were up to 23.30±0.20 mm against Streptococcus equi subsp.zooepidemicus ATCC35246.After eliminating the interference of organic acids and hydrogen peroxide,modified amino acids sequencing of the purified bacteriocin obtained by Q-Exactive,suggesting a lantibiotic.The bacteriocin,named suicin024,was heat,pH and protease resistant.The putative gene cluster involved in suicin024 produced by S.Suis CZJYW024 was amplified by PCR and sequence analysis revealed the presence of ten open reading frames named scz gene cluster.To assess the contribution of the gene cluster to antibacterial activity,the crucial scz cluster was deleted to generate the mutant strain ?scz.Consequently,the ?scz mutant strain lost the ability to produce bacteriocin compared with it's parental strain,suggesting that suicin024 was encoded by scz gene cluster.Given that S.suis CZJYW024 can not cause infections in a mouse model,and that it produces a bacteriocin with stable and antibacterial activity,it could potentially be used to prevent infections and to reduce antibiotic use in animal industry.Thus,they have been regarded as potential alternatives for conventional antibiotics,with promising applications in both the food and pharmaceutical industries.