| Streptococcus suis are worldwide zoonotic phathogens of many different diseases such as meningitis,septicemia,arthritis,endocarditis and may cause sudden death in swins or human,which made substantial economic losses for pig industry and became a substantial threat to human health especially those involved in the pig industry.So far, thrity-five different serotypes(type 1/2 and 1 through 34) of S.suis have been described.Of the 35 serotypes,Streptococcus suis type 2(SS2) is the most frequently isolated serotypes from pigs with disease,but strains belonging to other serotypes such as types 1/2,1,7,9 and 14 can also caused disease.However,not all SS2 strains are virulent,and there is variation in the virulence of those strains that are virulent.The trag and orf1616 was two of the novel infection-related factors,which were identified by in vivo induced antigen technology(IVIAT) from SS2 expression libraries with swine convalesecent sera in our former research.According to the sequence of trag of North American strain 89/1591 and the sequence of orf1616 of Europen strain P1/7,two pairs of primers were designed to detect the distribution of trag in total 43 SS isolates. Another pair of primers were designed to amplify the ORF of trag of 5 SS representive strains(ZY05719,HA9801,98012,SH040805,SH040917).Partial genes of trag and orf1616 were cloned and inserted into expression vector pET-28a(+) respectively,and induced by IPTG to express recombinant TRAG and ORF1616.The recombinant proteins were probed with swine convalescent sera and immune sera respectively.While the immunoreactivity were only presented with convalescent sera,which indicated that TRAG, ORF1616 might play a role during SS2 invasive course.A duplex PCR was developed for rapid and sensitive detection of virulent SS2 isolates. Two distinct DNA targets were based on the serotype 2(and 1/2) specific cps2j,and the newly infection-related gene orf1616.The sequence analysis of the PCR products and sensitivity and specificity test of the PCR assay were also done.The results showed that 31 isolates of virulent SS2 had amplified the two genes,but avirulent strain T15 only had cps2j.while cps2j and orf1616 had not amplified in 6 isolates of SS9 and 1 isolate of SS7, 1 isolate of SS1,1 isolate of SS1/2 and 2 isolates of group C streptococcal strains from pigs. The sequenced results of PCR production(cps2j,orf1616) templeted on Sichuan isolate ZY05719 were compared with correlated sequences cps2j(DQ207606),orf1616 (EF563980) in GenBank,the homology were 100%and 100%,repectively.The results showed the duplex PCR was a highly sensitive and specific tool,ideal for fast diagnosis and surveying epidemics.Two sets of oligonucleotide primers were designed and synthesized based on the published gene sequences of cps2j and orf1616 of SS2,and the loop-mediated isothermal amplification(LAMP) for detection of SS2 were developed after optimizing the factors. The LAMP reaction mix was optimized.The most optimal reaction temperature and time of the LAMP assay for the cps2j and orf1616 were 63℃,60℃and 60min,respectively.The cps2j were amplified in 32 SS2(32/32),orf1616 were amplified in 31 SS2 isolates,not in SS1,SS9,SS7 and other control bacteria strains.The results showed that the method was valid for detection of virulent isolates of SS2.The detection limit of this LAMP assay was around 110 fg of SS2 genomic DNA and 40 colonies forming units for pure cultures.These results suggested that detection of SS2 by LAMP was an effective and low-cost procedure with high specificity and sensitivity that required no specialized equipment.This assay was expected to become a valuable tool for rapid detection and identification of SS2. |
PDF Full Text Request