Establishment Of The PCR-ELISA Method For Detection Of Streptococcus Suis Ⅱ

Streptococcus suis II can cause meningitis and septicaemia in pigs. humans can be infected by this bacterialthrough certain ways. It is a significant pathogen of swine and human beings. In Jiang Su and Si Chuan provincesthere appeared human infections of the bacterial, which caused great casualty and huge economic loss. The detection methods for Streptococcus suis II were consist of microbe detection Method, serological detection method and molecular biology method at present. Microbe detection method made the initial detection of Streptococcus suis II according to cultural characteristics and biochemical characteristics of bacteria. However, the different and same serotype of Streptococcus suis II had diffierent biochemical reaction, so this method could not make a diagnosis. serological detection method detected samples according to capsular polysaccharide, some data showed that the different serotype had the same antigen and cross-reaction. Molecular biology detection method mainly included PCR, detected samples according to target band,but the results could not make accurate judgments. Therefore, to set up an extraordinary accurate and quick detection method for Streptococcus suis II,the PCR-ELISA were established.This experiment designed a pair of unique primer and probe according to the published serial number cps2j of the nucleotide by the capsular polysaccharide, the primer in the upstream marked the biotin, the primer in the downstream didnt mark anything. The probe was marked by the digoxin, and then extended and increased, and amplified the bacterial strain which was separated by the lab as the mold. Then, mixed the amplified product with the probe and in PCR hybridization would be carried on, the hybridizing temperature, time, and the nature alternation time and condition were optimized. Put the product of the hybridization into ELISA plate coated by Streptavidin, after throwing away the liquid and washing the board, and then put the antisubstance against the digoxin into it and make them react under the temperature of37℃. In this process, the two reaction times were optimized. And added OPD to develop color, and read the data through ELISA.The result of detecting the staphylococcus and other five Streptococcus suis Ⅱ through the optimized trial procedure, which indicated that this method had a good specificity. Diluted the bacterial liquid of842into1×100,1×101,1×102,1×103,1×104,1x105bacteria and then tested the sensitivity of the amplified product though the PCR reaction, the result of which showed that electrophoresis could detect100bacteria, however, PCR-ELISA detected10bacteria, which was ten times than the electrophoresis. Later we carried out a test toward the83clinical samples collected from the nasalpharynx, making a comparison between the PCR-ELISA and the PCR electrophoresis. Through the testing of the83clinical samples, the positive sample that PCR electrophoresis detected was17, positive rate was20%;while the PCR-ELISA detected was27, positive rate was32%. The test results showed that PCR-ELISA was optimized than the PCR electrophoresis for the clinical test. It was concluded that PCR-ELISA was a specific, sensitive, rapid and convenient method for detection of Streptococcus suis Ⅱ.