| Streptococcus suis(SS)is a Gram-positive bacterium widely distributed in nature world,a variety of animals such as pig,horse,cattle,sheep,chicken,and mouse can be infected by SS.Among which pig has the highest susceptibility,the infected pigs will develop clinical symptoms such as meningitis,arthritis,and sepsis.Human is also susceptible to SS,clinical symptoms include meningitis and sepsis.Due to the high mortality of swine streptococcicosis caused by SS and the rapid spread of the disease,it has brought huge economic losses to the pig industry and also seriously endangered human health.Therefore,rapid detection and diagnosis of swine streptococcicosis is the key to prevent the disease.Due to the complexity of the traditional pathogen isolation and culture and biochemical identification methods,it is difficult to make a timely and accurate diagnosis of the disease,and it is not suitable for the detection of a large number of samples.Therefore,establishment of a rapid,accurate and efficient detection method is of great significance for the prevention of the disease and epidemiological research.Muramidase-released protein(Mrp)is an important virulence marker factor of SS,which has good immunogenicity.In this study,the segment from 2617 to 3710 bp of mrp gene was amplified,and was further prokaryotic expressed and purified.Western blot analysis of the purified Mrp protein showed that the protein has good immunogenicity.Using the purified Mrp protein as the coating antigen,an indirect ELISA diagnostic method for detecting SS serum antibodies was established.The experiments were optimized for the antigen coating concentration,the dilution fold and incubation time of the serum,the blocking solution and the blocking temperature and time,the dilution fold and incubation time of the secondary antibody,and the color developing time of the substrate.Finally,the best experimental conditions for this indirect ELISA method were determined: the antigen coating concentration was 0.05 ?g / mL;the dilution fold of serum was 1: 400,the incubation temperature was 37?,and the incubation time was 30 minutes;5% skim milk was used as the blocking solution which blocked at 37? for 2 h;The secondary antibody was diluted to 1: 15000 and incubated at 37? for 30 minutes;The color developing time of substrate was 15 minutes.The threshold value was determined at 0.226.The specificity,sensitivity,repeatability,and stability of the method were evaluated afterwards.Then it was used to detection of the clinical samples.The results showed that the specificity of this method was 97%,and there was no cross reaction with the positive sera of several common pathogens of swine,such as haemophilus parasuis(HPS),actinobacillus pleuropneumoniae(APP),porcine reproductive and respiratory syndrome virus(PRRSV),etc.Meanwhile,the positive sera of streptococcus suis type 2(SS2),type 7(SS7),type 9(SS9)and type 12(SS12)were all showed positive reaction.The sensitivity was 96%,and the minimum detection limit of the serum sample was 1: 3200-fold dilution.For the repeatability test,the maximum variation coefficients of the intra-batch repeatability test and inter-batch repeatability test were 4.70% and 7.20%,respectively.The indirect ELISA method and slide agglutination test were used to detect 100 clinical serum samples at the same time,it showed that the coincidence rate was 85.3%;440 clinical pig serum samples were clinical pig serum samples were tested by the indirect ELISA method,and the positive detection rate was 27.7%;The variation coefficients of specificity and sensitivity of 96-well plate coated with antigen were less than 10% within 6 months of storage at 4?.The total results showed that the indirect ELISA established in this study has good specificity,sensitivity,repeatability and stability,which is suitable for the detection of serum antibodies to SS2,SS7,SS9,and SS12.It is expected to provide a reliable technical means for the diagnosis and epidemiological investigation of swine streptococcicosis. |
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