Establishment of Real-time Fluorescence PCR to Detect Streptococcus suis Serotype 2
LUO Bao-zheng;BO Qing-ru;CHEN Jing-fan;XU Hai-nie;YANG Su;YANG Jian-yun; SHA Cai-hua;LIAO Xiu-yun
1. Zhuhai Entry-exit Inspection and Quarantine Bureau，Zhuhai 519015, China; 2. College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
Abstract：A real-time fluorescence polymerase chain reaction(PCR) method to detect Streptococcus suis serotype 2 was established. Primers and Taqman probe were designed according to cps2I(capsular polysaccharide 2I) gene with software Primer Express 2.0 and Oligo6.0. Eighty-one base pair DNA fragment was amplified from S. suis serotype 2 genome DNA, and the PCR product was cloned into pMD18-T vector. Sequencing result confirmed that DNA fragment was target segment. Real-time fluorescence PCR amplification curve on Lightcycler showed that this method could successfully amplify S. suis serotype 2, while reference S. suis strain, E. coli, Salmonella, Staphylococcus aureu, Shigella, Listeria monocytoge and blank control were all negative, so it had good specificity. Ten-fold dilution of S. suis serotype 2 was used to measure the sensitivity of real-time fluorescence PCR. Ten bacteria could be detected in one PCR reaction and the reaction could be completed within 30 min. To examine the stability of the real-time fluorescence PCR, positive control was detected at two different times with 20 repeats. Result showed that Ct value of two times had no statistic difference(P > 0.05), thus this method has a reliable stability. The newly-built real-time fluorescence PCR has potential to apply in entry-exit inspection and quarantine.
